Mol Microbiol, 1989 Jul, 3(7), 903 - 10 Cloning and characterization of an Escherichia coli gene, pcnB, affecting plasmid copy number; March JB et al.; A gene, pcnB, affecting the copy number of ColE1-related plasmids has been cloned and mapped to 3.6 min on the Escherichia coli chromosome between panD and fhu . The gene encodes a previously undescribed 48 kD protein . Several independently isolated mutants exhibiting the same phenotype, reduced copy number, have been shown to be pcnB-. Mol Microbiol, 1989 Jul, 3(7), 891 - 902 Assembly of colicin genes from a few DNA fragments . Nucleotide sequence of colicin D; Roos U et al.; The nucleotide sequence of a 2.4 kb Dral-EcoRV fragment of pColD-CA23 DNA was determined . The segment of DNA contained the colicin D structural gene (cda) and the colicin D immunity gene (cdi) . From the nucleotide sequence it was deduced that colicin D had a molecular weight of 74,683 D and that the immunity protein had a molecular weight of 10,057 D . The amino-terminal portion of colicin D was found to be 96% homologous with the same region of colicin B . Both colicins share the same cell-surface receptor, FepA, and require the TonB protein for uptake . A putative TonB box pentapeptide sequence was identified in the amino terminus of the colicin D protein sequence . Since colicin D inhibits protein synthesis, it was unexpected that no homology was found between the carboxy-terminal part of this colicin and that of the protein synthesis inhibiting colicin E3 and cloacin DF13 . This could indicate that colicin D does not function in the same manner as the latter two bacteriocins . The observed homology with colicin B supports the domain structure concept of colicin organization . The structural organization of the colicin operon is discussed . The extensive amino-terminal homology between colicins D and B, and the strong carboxy-terminal homology between colicins B, A, and N suggest an evolutionary assembly of colicin genes from a few DNA fragments which encode the functional domains responsible for colicin activity and uptake. Mol Microbiol, 1989 Jul, 3(7), 869 - 78 Molecular genetic analysis of FNR-dependent promoters; Eiglmeier K et al.; In enteric bacteria, the expression of many genes encoding various anaerobic electron transfer functions is controlled by FNR, the product of the autoregulated fnr gene . FNR is structurally and functionally homologous to CAP, the catabolite gene activator protein, and increased FNR production strongly stimulates transcription of its target genes . By analysis of RNA produced in vivo the promoters of four FNR-dependent genes were localized and shown to display a common arrangement . A 22bp dyad symmetry was found about 30 nucleotides upstream of the transcriptional startpoints and a similar sequence was shown to overlap the site of transcription initiation in the negatively controlled fnr gene . The consensus sequence for the half site recognized by FNR (AAA-TTGAT) is only slightly different from that of CAP (AA-TGTGA) . Studies with two mutant frd promoters from Escherichia coli, displaying altered regulation and FNR response, provided additional evidence for recognition of this sequence by FNR. FEMS Microbiol Lett, 1989 Jul 1, 51(1), 79 - 83 Comparison of R- and S-form lipopolysaccharides fractionated from Escherichia coli UKT-B lipopolysaccharide in pyrogen and Limulus tests; Komuro T et al.; Different LPS was shown to have a relatively different proportion of O-specific chain-less (R-form) LPS by polyacrylamide gel electrophoresis (PAGE) with sodium deoxycholate (DOC) . By using DOC-PAGE, S-form LPS having O-chain with approximately 11 repeating units on average (S-Fr) and O-chain-less LPS (R-Fr) were separated from Escherichia coli UKT-B S-form LPS . Significantly stronger pyrogenicity was observed in R-Fr than in S-Fr when measured on the weight basis . Similar result was observed in Limulus test . Comparing biological activities of different S-form LPS, attention should be given to the amounts of co-existing R-form LPS. FEMS Microbiol Lett, 1989 Jul 1, 51(1), 11 - 4 Interactions of membrane lipoproteins with the murein sacculus of Escherichia coli as shown by chemical crosslinking studies of intact cells; Leduc M et al.; Proteins that were closely associated with murein in intact cells of Escherichia coli were studied by treating {3H}leucine and {3H}palmitate-labeled cells with the chemical crosslinking reagent dithiobis(succinimidylpropionate) . Murein was purified and crosslinked peptides were released from the murein by treatment with beta-mercaptoethanol . Nine murein-associated {3H}leucine-labeled peptides were identified . Five of the nine peptides were lipoproteins, based on labeling with {3H}palmitate, protease sensitivity and gel electrophoretic correspondence to membrane lipoproteins present in uncrosslinked cell envelope preparations . The results suggest that these membrane lipoproteins may play a significant role in the structural integration of the murein and membrane layers of the cell envelope. EMBO J, 1989 Jul, 8(7), 2095 - 9 Positive charges in the cytoplasmic domain of Escherichia coli leader peptidase prevent an apolar domain from functioning as a signal; Laws JK et al.; Leader peptidase, an integral transmembrane protein of Escherichia coli, requires two apolar topogenic elements for its membrane assembly: a 'hydrophobic helper' and an internal signal . The highly basic cytoplasmic region between these domains is a translocation poison sequence, which we have shown blocks the function of a preceding signal sequence . We have used oligonucleotide-directed mutagenesis to remove positively charged residues within this polar domain to determine if it is the basic character in this region that has the negative effect on translocation . Our results show that mutations that remove two or more of the positively charged residues within the polar region no longer block membrane assembly of leader peptidase . In addition, when the translocation poison domain (residues 30-52) is replaced with six lysine residues, the preceding apolar domain cannot function as an export signal, whereas it can with six glutamic acids . Thus, positively charged residues within membrane proteins may have a major role in determining the function of hydrophobic domains in membrane assembly. Curr Genet, 1989 Jul, 16(1), 21 - 5 A novel class of vector for yeast transformation; Chinery SA et al.; We have constructed a set of hybrid yeast Escherichia coli vectors which utilise the site specific recombination function of the Saccharomyces cerevisiae 2 microns plasmid to completely eliminate the bacterial moiety upon introduction into yeast . A number of these plasmids have been shown to exhibit high inheritable stability in both laboratory and industrial strains during non-selective growth . These plasmids are beneficial for the genetic modification of industrial yeast, particularly those used in the production of food and beverages, and are of benefit in the study of plasmid maintenance and heterologous gene expression. Cesk Epidemiol Mikrobiol Imunol, 1989 Jul, 38(4), 204 - 8 {Evaluation of phagocytosis of alpha hemolytic strains of Escherichia coli using the fluorescence method}; Siegfried L et al.; The authors investigated phagocytosis in 37 alpha haemolytic strains of E . coli by the fluorescent method, after opsonization of bacteria with 5% and concentrated human serum . The strains were isolated from infants with gastrointestinal and extraintestinal infections and belonged to eleven serogroups . During opsonization of the strain with concentrated serum, as compared with opsonization of strains with 5% serum, higher rates of killing were recorded . As compared with a control strain of E . coli K12 after opsonization with concentrated serum in the examined strains the value of killing was on average by 20% lower . Differences in the killing of examined strains were recorded also within a single serogroup which may be ascribed to the different provision of strains with plasmids. Cell Biol Int Rep, 1989 Jul, 13(7), 619 - 24 Estrogen-induced expression of mouse lactate dehydrogenase-A gene; Li SS et al.; The synthesis of lactate dehydrogenase-A isoenzyme was shown to increase significantly in the uterus of immature mice treated by diethylstilbestrol . The expression of the mouse LDH-A promoter and cat fusion gene in Chinese hamster ovary cells was also induced by 17 beta-estradiol and diethylstilbestrol. Radiobiologiia, 1989 Jul-Aug, 29(4), 495 - 500 {The limit of the modifiability of cellular radiosensitivity in sequential exposure to ionizing radiation and physical factors of a nonionizing nature}; Averin VI et al.; The application of a mathematical model of synergism in describing the consecutive combined actions of ionizing radiation and other physical agents has been considered . Using various cell systems it has been shown that the model permits to predict the highest dose modifying factor and conditions in which it can be achieved. Mol Cell Biol, 1989 Jul, 9(7), 3058 - 72 Dual bidirectional promoters at the mouse dhfr locus: cloning and characterization of two mRNA classes of the divergently transcribed Rep-1 gene; Linton JP et al.; The mouse dihydrofolate reductase gene (dhfr) is a housekeeping gene expressed under the control of a promoter region embedded in a CpG island--a region rich in unmethylated CpG dinucleotides . A divergent transcription unit exists immediately upstream of the dhfr gene which is coamplified with dhfr in some but not all methotrexate-resistant cell lines . We show that the promoter region for this gene pair consists of two bidirectional promoters, a major and minor promoter, which are situated within a 660-base-pair region upstream of the dhfr ATG translation initiation codon . The major promoter controls over 90% of dhfr transcription, while the minor promoter directs the transcription of the remaining dhfr mRNAs . The major promoter functions bidirectionally, transcribing a divergent 4.0-kilobase poly(A) mRNA (class A) in the direction opposite that of dhfr transcription . The predicted protein product of this mRNA is 105 kilodaltons . The minor promoter also functions bidirectionally, directing the transcription of at least two divergent RNAs (class B) . These RNAs, present in quantities approximately 1/10 to 1/50 that of the class A mRNAs, are 4.4- and 1.6-kilobase poly(A) mRNAs . cDNAs representing both class A and class B mRNAs have been cloned from a mouse fibroblast cell line which has amplified the dhfr locus (3T3R500) . DNA sequence analysis of these cDNAs reveals that the class A and class B mRNAs share, for the most part, the same exons . On the basis of S1 nuclease protection analysis of RNA preparations from several mouse tissues, both dhfr and divergent genes showed similar levels of expression but did show some specificity in start site utilization . Computer homology searches have revealed sequence similarity of the divergent transcripts with bacterial genes involved in DNA mismatch repair, and we therefore have named the divergently transcribed gene Rep-1. Mol Cell Biol, 1989 Jul, 9(7), 3000 - 8 SSC1, an essential member of the yeast HSP70 multigene family, encodes a mitochondrial protein; Craig EA et al.; SSC1 is an essential member of the yeast HSP70 multigene family (E . Craig, J . Kramer, and J . Kosic-Smithers, Proc . Natl . Acad . Sci . USA 84:4156-4160, 1987) . Analysis of the SSC1 DNA sequence revealed that it could encode a 70,627-dalton protein that is more similar to DnaK, an Escherichia coli hsp70 protein, than other yeast hsp70s whose sequences have been determined . Ssc1p was found to have an amino-terminal extension of 28 amino acids, in comparison with either Ssa1p, another hsp70 yeast protein, or Dnak . This putative leader is rich in basic and hydroxyl amino acids, characteristic of many mitochondrial leader sequences . Ssc1p that was synthesized in vitro could be imported into mitochondria and was cleaved in the process . The imported protein comigrated with an abundant mitochondrial protein that reacted with hsp70-specific antibodies . We conclude that Ssc1p is a mitochondrial protein and that hsp70 proteins perform functions in many compartments of the cell. Mol Cell Biol, 1989 Jul, 9(7), 2944 - 9 Defining the sequence specificity of DNA-binding proteins by selecting binding sites from random-sequence oligonucleotides: analysis of yeast GCN4 protein; Oliphant AR et al.; We describe a new method for accurately defining the sequence recognition properties of DNA-binding proteins by selecting high-affinity binding sites from random-sequence DNA . The yeast transcriptional activator protein GCN4 was coupled to a Sepharose column, and binding sites were isolated by passing short, random-sequence oligonucleotides over the column and eluting them with increasing salt concentrations . Of 43 specifically bound oligonucleotides, 40 contained the symmetric sequence TGA(C/G)TCA, whereas the other 3 contained sequences matching six of these seven bases . The extreme preference for this 7-base-pair sequence suggests that each position directly contacts GCN4 . The three nucleotide positions on each side of this core heptanucleotide also showed sequence preferences, indicating their effect on GCN4 binding . Interestingly, deviations in the core and a stronger sequence preference in the flanking region were found on one side of the central C . G base pair . Although GCN4 binds as a dimer, this asymmetry supports a model in which interactions on each side of the binding site are not equivalent . The random selection method should prove generally useful for defining the specificities of other DNA-binding proteins and for identifying putative target sequences from genomic DNA. Mol Cell Biol, 1989 Jul, 9(7), 2854 - 9 RNA polymerase II subunit RPB4 is essential for high- and low-temperature yeast cell growth; Woychik NA et al.; RPB4 encodes the fourth-largest RNA polymerase II subunit in Saccharomyces cerevisiae . The RPB4 gene was cloned and sequenced, and its identity was confirmed by amino acid sequence analysis of tryptic peptides from the purified subunit . The RPB4 DNA sequence predicted a protein of 221 amino acids with a molecular mass of 25,414 daltons . The central 100 amino acids of the RPB4 protein were found to be similar to a segment of the major sigma subunit in Escherichia coli RNA polymerase . Deletion of RPB4 produced cells that were heat and cold sensitive but could grow, albeit slowly, at intermediate temperatures . RNA polymerase II lacking the RPB4 subunit exhibited markedly reduced activity in crude extracts in vitro . The RPB4 subunit, although not essential for mRNA synthesis or enzyme assembly, was essential for normal levels of RNA polymerase II activity and indispensable for cell viability over a wide temperature range. Mutagenesis, 1989 Jul, 4(4), 254 - 8 DNA adduct formation by 7-methoxy-2-nitro-naphtho{2,1-b}furan (R7000), an extremely potent mutagen; Touati E et al.; The effects on DNA, in bacteria, of 7-methoxy-2-nitro-naphtho{2,1-b}furan (R7000), a very potent genotoxic product from the 2-nitronaphthofuran series, were investigated with two different approaches: (i) measurement of the binding of the radiolabelled mutagen to DNA and (ii) detection by the '32P-postlabelling' method of DNA adducts following treatment with unlabelled mutagens . The covalent binding of R7000 to DNA in Escherichia coli was demonstrated by both methods, and in the latter case it was found to involve the formation of nine different adducts . Formation of adducts by R7000 was shown to require metabolic activation of the compound. Indian J Pathol Microbiol, 1989 Jul, 32(3), 198 - 206 Studies on the pathogenicity of classical enteropathogenic Escherichia coli strains isolated from acute diarrhoea among children 0-5 years of age; Ananthan S et al.; The mechanisms of pathogenicity in EPEC strains were studied in tissue culture . Escherichia coli was isolated as the predominant organism in the primary culture of 1293 (70.54%) diarrhoeal cases . 284 (90.44%) cases from the age group of 1-6 months showed Escherichia coli as the predominant organism . Classical Enteropathogenic Escherichia coli (EPEC) were detected in 311 (24.05%) cases . Among EPEC isolates 277 (89.06%) did not produce either LT or ST . 32 (10.28%) produced LT or ST . 2 strains produced verotoxins belong to serotypes 0:86; K:61, 0.26; K:60, serogroups 0.86:K61 0142:K86, 0:128:K67, 0 126: 71, 0125:K 70, 0119: k69 showed localised adherence and serogroups 0111:K58-055:K59 showed both localised and diffused adherence to HeLa cells. Indian J Pathol Microbiol, 1989 Jul, 32(3), 161 - 6 Drug resistance and colicin production among various serotypes of Escherichia coli of animal origin; Singh M et al.; One hundred and twenty two strains of E . coli isolated from clinical conditions of animals were studied for the production of colicins and their sensitivity behaviour towards 16 antibiotics and trimethoprim-sulphamethoxazole . Out of 122 strains, 27 (22.1%) were found colicinogenic and 25 (93%) of these colicinogenic strains were found resistant to one or more of drugs in various combinations . All the 27 colicinogenic strains could be typed into 15 serogroups . Serogroup 084 was found predominant . Transfer of R-plasmids and Col-plasmids were observed in 5 (18.5%) strains individually . In one strain both drug resistance and colicin production determinants were transferred enblock . Such transconjugants will be more invasive and virulent and will create serious chemotherapeutic problems. Genes Dev, 1989 Jul, 3(7), 921 - 35 A protein involved in minichromosome maintenance in yeast binds a transcriptional enhancer conserved in eukaryotes; Passmore S et al.; The Saccharomyces cerevisiae MCM1 gene product is a protein with multiple functions . It is a transcription factor necessary for expression of mating-type-specific genes and is also required for the maintenance of minichromosomes . MCM1 shows DNA-binding specificities similar to those of two previously reported DNA-binding factors, pheromone/receptor transcription factor (PRTF) and general regulator of mating type (GRM); like PRTF, its activity can be modulated by the alpha 1 protein . MCM1 binds to the dyad symmetry element 5'-CCTAATTAGG and related sequences, which we refer to as MCM1 control elements (MCEs) . MCEs are found within the regulatory regions of a- and alpha-specific genes . Direct and indirect DNA binding assays suggest that a conserved 5'-ATTAGG in one-half of the dyad symmetry element is important for MCM1 binding whereas variants in the other half are tolerated . We have used a novel DNase I 'nicking interference' assay to investigate the interaction of MCM1 with its substrate . These data suggest that MCM1 binds as a dimer, interacting symmetrically with the ATTAGG residues in each half of the binding site . MCM1 contains striking homology to the DNA-binding domain of the human serum response factor (SRF) which mediates the transient transcriptional activation of growth-stimulated genes by binding to the serum response element (SRE) . We have shown that MCM1 binds to the human c-fos SRE in vitro and, like other MCEs, the c-fos SRE exhibits MCM1-mediated upstream activating sequence (UAS) activity in vivo. Genes Dev, 1989 Jul, 3(7), 1045 - 52 New suppressors of signal-sequence mutations, prlG, are linked tightly to the secE gene of Escherichia coli; Stader J et al.; Analysis of more than 100 extragenic suppressors of the lamB14D signal-sequence mutation (changes Val in the hydrophobic core region at position 14 to Asp) has revealed alterations that appear to lie at prlA (secY) and secA (prlD), two loci known to be mutable to suppressor alleles, and a new suppressor termed prlG . One allele of the new suppressor class, prlG1, has been characterized in some detail . This suppressor counteracts, to some degree, the export defect conferred by a variety of signal-sequence mutations in two different genes, lamB and malE . Genetic analysis shows that the dominant suppressor mutations are linked tightly to, and probably allelic with, the gene secE . This result, coupled with data obtained with conditional-lethal alleles of secE, argues strongly that SecE is an important component of the cellular protein export machinery in Escherichia coli. Genes Dev, 1989 Jul, 3(7), 1035 - 44 The secE gene encodes an integral membrane protein required for protein export in Escherichia coli; Schatz PJ et al.; Genetic screening and selection procedures employing a secA-lacZ fusion strain repeatedly have yielded mutations in four genes affecting the protein export pathway of Escherichia coli . These genes are secA, secD, prlA/secY, and secE . We discuss the significance of the failure to find new sec genes after extensive use of this approach . One of the genes, secE, has been characterized in some detail . From the DNA sequence of the gene and analysis of alkaline phosphatase fusions to the SecE protein, we propose that it is a 13,600-dalton integral cytoplasmic membrane protein . The data presented here and in the accompanying paper strongly suggest that secE has an important role in E . coli protein export. DNA, 1989 Jul-Aug, 8(6), 429 - 36 Production of human recombinant proapolipoprotein A-I in Escherichia coli: purification and biochemical characterization; Moguilevsky N et al.; A human liver cDNA library was used to isolate a clone coding for apolipoprotein A-I (Apo A-I) . The clone carries the sequence for the prepeptide (18 amino acids), the propeptide (6 amino acids), and the mature protein (243 amino acids) . A coding cassette for the proapo A-I molecule was reconstructed by fusing synthetic sequences, chosen to optimize expression and specifying the amino-terminal methionine and amino acids -6 to +14, to a large fragment of the cDNA coding for amino acids 15-243 . The module was expressed in pOTS-Nco, an Escherichia coli expression vector carrying the regulatable lambda PL promoter, leading to the production of proapolipoprotein A-I at up to 10% of total soluble proteins . The recombinant polypeptide was purified and characterized in terms of apparent molecular mass, isoelectric point, and by both chemical and enzymatic peptide mapping . In addition, it was assayed in vitro for the stimulation of the enzyme lecithin: cholesterol acyltransferase . The data show for the first time that proapo A-I can be produced efficiently in E . coli as a stable and undegraded protein having physical and functional properties indistinguishable from those of the natural product. Zentralbl Bakteriol, 1989 Jul, 271(2), 205 - 13 Role of alpha-hemolysin for the in vitro phagocytosis and intracellular killing of Escherichia coli; Gadeberg OV et al.; The role of alpha-hemolysin for the elimination of Escherichia coli by phagocytes in vitro was investigated using sets of isogenic strains which included wild-type alpha-hemolytic strains, derived strains with a reduced production of alpha-hemolysin and derived nonhemolytic strains . Phagocytosis and intracellular killing of the bacteria by human blood granulocytes or monocytes were measured using growth inhibition techniques . alpha-hemolytic strains were phagocytosed and killed to a lesser extent than isogenic strains with a reduced production of alpha-hemolysin and isogenic nonhemolytic strains . The results obtained with granulocytes were similar to those obtained with monocytes although the elimination of bacteria by monocytes was less than that by granulocytes . These results strongly suggest that production of alpha-hemolysin is a means by which E . coli counteracts the activity of phagocytes by injuring these cells with the toxin. Z Naturforsch {C}, 1989 Jul-Aug, 44(7-8), 715 - 8 Deoxyribonucleotide synthesis in an Escherichia coli mutant (H 1491) which lacks ribonucleotide reductase subunit B2; Harder J et al.; An iron-sensitive mutant of E . coli with a Mudl phage insertion in the nrdB gene lacks subunit B2 of the key enzyme of DNA synthesis, ribonucleotide reductase . Nevertheless, these cells are capable of growing in minimal media under anaerobic conditions, indicating a second enzyme or pathway for deoxyribonucleotide synthesis . We here show that ribonucleotide reduction cannot be unambiguously measured in bacterial extracts whereas phosphorylase-catalyzed deoxyribosyl transfer does occur; however these salvage reactions could not function in vivo in the absence of deoxyribosides . It is suggested that the cells possess a specific, anaerobic ribonucleotide reductase which escapes detection under aerobic standard conditions, similar to the situation found in strictly anaerobic methanogens. Biochem J, 1989 Jul 1, 261(1), 301 - 4 Evidence for an arginine residue at the coenzyme-binding site of Escherichia coli isocitrate dehydrogenase; McKee JS et al.; The arginine-specific reagent phenylglyoxal inactivated the active, dephosphorylated, form of Escherichia coli isocitrate dehydrogenase rapidly in a pseudo-first-order process . Both NADP+ and NADPH protected the enzyme against inactivation . Phenylglyoxal appeared to react with one arginine residue per subunit, and the extent of the reaction was proportional to the extent of the inactivation . In contrast, the phosphorylated form of isocitrate dehydrogenase did not react detectably with phenylglyoxal . The data indicate that the coenzyme-binding site of isocitrate dehydrogenase contains a reactive arginine residue that is protected by phosphorylation, and are consistent with the hypothesis that phosphorylation of the enzyme occurs close to or at its active site. Avian Dis, 1989 Jul-Sep, 33(3), 502 - 5 Congo red binding by Escherichia coli isolates from chickens; Yoder HW Jr; An attempt was made to use a recently reported special Congo red medium to determine the pathogenicity of Escherichia coli isolates obtained from chickens . The inclusion of bile salts in the Congo red medium as described in previous reports by others was found in the current experiments to cause the production of red colonies from almost all E . coli cultures tested, including known Congo red-negative control cultures . Cultures of E . coli, regardless of their pathogenic history, rarely produced red colonies on the Congo red medium without added bile salts . Numerous isolates of other bacterial genera were examined and found to produce red colonies on the Congo red medium with or without added bile salts. Avian Dis, 1989 Jul-Sep, 33(3), 473 - 8 Experimental reproduction of airsacculitis and septicemia by aerosol exposure of 1-day-old chicks using Congo red-positive Escherichia coli; Gjessing KM et al.; To further demonstrate the association of phenotype and virulence of Congo red Escherichia coli (CREC), experiments were designed to reproduce airsacculitis and colisepticemia in 1-day-old chicks via aerosol exposure . In eight separate experiments in which a total of 462 chicks were exposed to CREC, the mortality rate was 4.11% and the morbidity rate was 13.4%, with both the dead and diseased chicks showing lesions of fibrinous airsacculitis, pericarditis, and perihepatitis . In contrast, when the corresponding non-CREC derivatives (the same E . coli strains, but not expressing the CR phenotype) were used as the aerosol inoculum in five additional experiments using 284 chicks, no chicks died and no lesions were observed . These results clearly show a strong correlation between the expression of the CR phenotype and virulence in avian E . coli. Am J Vet Res, 1989 Jul, 50(7), 1029 - 36 Pathogenicity of Escherichia coli O115:K"V165" strains isolated from pigs with diarrhea; Fairbrother JM et al.; Eighteen strains of Escherichia coli serogroup O115:K"V165" isolated from 1- to 8-week-old pigs with diarrhea were tested for toxigenicity, pathogenicity in pigs and mice, serum resistance, mannose-resistant hemagglutination (MRHA), F165 and other surface antigens, colicin V (Col V), aerobactin, and biotype . Twelve strains were positive for heat-stable enterotoxin (STb), MRHA-negative, and F165-negative; 5 strains were enterotoxin-negative, MRHA-positive, and F165-positive; and 1 strain was MRHA-positive, but F165- and enterotoxin-negative . Six of the 12 STb-positive strains moderately colonized the ileum of newborn colostrum-deprived pigs within 24 hours after inoculation . Two of the colonizing strains were able to induce watery diarrhea . All 12 STb-positive strains were nonpathogenic for adult mice and were serum-sensitive; 11 of 12 were Col V-negative, 9 of 12 did not produce aerobactin, and 10 of 12 belonged to biotypes other than 1 or 2 . All 6 enterotoxin-negative strains colonized the small and large intestines, associated with peritoneal serosal surfaces, and induced septicemia and polyserositis in newborn colostrum-deprived pigs 1 to 2 days after inoculation . In contrast, 3 STb-positive strains poorly colonized the intestines and did not induce septicemia in pigs at 3 days after inoculation . All 6 enterotoxin-negative strains were Col V-positive, produced aerobactin, and belonged to biotype 1 or 2 . Of the 5 enterotoxin-negative, F165-positive strains, only 4 were pathogenic for intraperitoneally inoculated adult mice and were serum-resistant . The enterotoxin-negative, F165-negative strain was neither serum-resistant nor mouse-pathogenic.(ABSTRACT TRUNCATED AT 250 WORDS) Res Vet Sci, 1989 Jul, 47(1), 119 - 24 Biochemical and haematological evidence of endotoxic shock in gnotobiotic lambs with watery mouth disease; Hodgson JC et al.; Eight gnotobiotic lambs deprived of colostrum were infected by mouth when two hours old with nonenterotoxigenic strains of Escherichia coli . All developed clinical signs of watery mouth disease and seven died within 24 hours . The mean concentrations of several blood constituents were determined in samples taken at intervals until 24 hours after infection in infected lambs and in four control lambs . The biochemical and haematological changes observed in the lambs developing watery mouth disease were those characteristic of endotoxic shock. J Clin Microbiol, 1989 Jul, 27(7), 1697 - 9 Identification of Escherichia coli that produces heat-stable enterotoxin STA by a commercially available enzyme-linked immunoassay and comparison of the assay with infant mouse and DNA probe tests; Scotland SM et al.; By using the infant mouse test and a commercially available competitive enzyme-linked immunosorbent assay (ELISA), 100 strains of Escherichia coli carrying STA1 or STA2 genes were shown to produce the heat-stable enterotoxin STA . An additional 100 strains were negative in both tests . The ELISA was easy to perform, and results were available within 24 h . Testing strains with an enzyme-linked DNA probe kit that incorporated both STA1- and STA2-specific oligonucleotides showed that the 100 strains positive in the mouse test and ELISA also hybridized with the mixed probe . Two strains carrying STA1 genes but negative in the mouse test and ELISA also hybridized with the mixed probe. J Clin Microbiol, 1989 Jul, 27(7), 1684 - 8 Simultaneous detection of Escherichia coli heat-stable and heat-labile enterotoxin genes with a single RNA probe; Saez-Llorens X et al.; A single RNA probe was synthesized and used to detect simultaneously the methanol-soluble heat-stable enterotoxin and heat-labile enterotoxin genes in Escherichia coli strains . The results with the biotinylated or radioactive probe correlated 100% with the biological assay results for both toxins . The RNA probe detected the three known heat-stable enterotoxin A alleles. J Clin Microbiol, 1989 Jul, 27(7), 1617 - 22 Sensitive receptor-specified enzyme-linked immunosorbent assay for Escherichia coli verocytotoxin; Basta M et al.; The specific identification of verocytotoxin (VT)-producing Escherichia coli (VTEC) requires the detection of VTs in bacterial culture filtrates or the detection of genes encoding these toxins in bacterial cells by specific DNA probes . The standard method for detecting these toxins involves a time-consuming, labor-intensive, and expensive cytotoxicity assay . We have developed a specific, highly sensitive receptor-specified enzyme-linked immunosorbent assay (RELISA) to detect VT1, one of at least two VTs implicated in human disease . The assay is based on the affinity of VT1 for the glycolipid globotriosyl ceramide (Gb3) . Gb3 was de-N-acylated to yield lyso-Gb3, which is more polar but retains VT1 binding . Lyso-Gb3 was used to sensitize microdilution plates to bind VT1 for subsequent immunodetection . This RELISA was used to detect VT1 in the culture supernatant of a variety of bacteria of known VT status . The assay was compared with the highly sensitive cell cytotoxicity assay for their abilities to detect VT . The RELISA was as sensitive as the cytotoxicity assay and, in a blind study, 100% specific . This assay will provide a quick, specific, efficient adjunct to the diagnosis and epidemiological study of VTEC infections and their relationship to human disease. Can J Vet Res, 1989 Jul, 53(3), 306 - 12 Effects of Escherichia coli Shiga-like toxins (verotoxins) in pigs; Gannon VP et al.; Escherichia coli K12 strains TB1(pCG5), with the genes for Shiga-like toxin IIv from an edema disease isolate of E . coli and TB1(pCG5-1), with the toxin genes inactivated by transposon mutagenesis, were used to test the hypothesis that Shiga-like toxin IIv was the same as edema disease principle . Ammonium sulfate precipitated culture supernatants from the pair of E . coli K12 strains and from a wild edema disease isolate of E . coli (E145) were tested for their ability to induce signs and lesions of edema disease in intravenously inoculated weaned pigs . Similar preparations from E . coli which produce Shiga-like toxins I and II were also tested . Preparations from E . coli TB1 (pCG5) and E145 contained high levels of Shiga-like toxin IIv and induced signs and lesions similar to those seen in edema disease, whereas preparations from E . coli TB1 (pCG5-1) failed to induce signs or lesions of edema disease . All Shiga-like toxin preparations produced delayed neurological signs, fibrinoid necrosis of arterioles and hemorrhages in the cerebellum of pigs . High doses of Shiga-like toxin IIv were associated with superficial necrosis of the colonic epithelium and vasculitis . Shiga-like toxins I and II resulted in kidney lesions but no enteric pathology . Shiga-like toxin II preparations had the lowest median lethal dose for pigs, Shiga-like toxin IIv was intermediate and Shiga-like toxin I was the least toxic. J Clin Pathol, 1989 Jul, 42(7), 755 - 8 Comparison of Y1 mouse adrenal cell and coagglutination assays for detection of Escherichia coli heat labile enterotoxin; Chapman PA et al.; A commercial coagglutination assay (COA; Phadebact LT-ETEC) was compared with a Y1 mouse adrenal cell assay for detecting the heat labile enterotoxin of Escherichia coli . Of four different media evaluated for use with the COA, only one (modified blood agar) gave a positive result with all strains known to produce heat labile enterotoxin . With modified blood agar, the COA detected 74 (85%) of 87 such strains . Eighty six strains negative by cell culture assay were also negative by COA, and one strain positive by COA could not be confirmed by cell culture . The Phadebact LT-ETEC kit provides a simple, sensitive, and economical method for detecting E coli heat labile enterotoxin. Eur J Biochem, 1989 Jul 1, 182(3), 501 - 6 Alternative occupancy of a dual ribosomal binding site by mRNA affected by translation initiation factors; Canonaco MA et al.; The interaction between Escherichia coli 30S ribosomal subunits and mRNAs, and the effect of the initiation factors on this process, have been studied using MS2 RNA, polyribonucleotides and model mRNAs encoded by synthetic genes . The interactions were analyzed by gel filtration, by sucrose gradient centrifugation and by competition for ribosome binding between the various mRNAs and a Shine-Dalgarno deoxyoctanucleotide . It was found that the initiation factors do not significantly affect the Shine-Dalgarno interaction nor the apparent Ka values of the 30S-subunit-mRNA binary complexes, but influence the positioning of the mRNAs on the 30S subunit with respect to the Shine-Dalgarno octanucleotide . The results suggest that, in the absence of initiation factors, the mRNA occupies a ribosomal "stand-by" site which is close to or includes the region where the Shine-Dalgarno interaction takes place; in the presence of the factors, the mRNA is shifted away from the stand-by site, towards another ribosomal site with similar affinity for the mRNA . This shift does not require the presence of fMet-tRNA and, depending upon the type of mRNA, is mediated by IF-2 and/or IF-3. Proc Natl Acad Sci U S A, 1989 Jul, 86(14), 5492 - 6 UV induction of coliphage 186: prophage induction as an SOS function; Lamont I et al.; Our results show that UV induction of the 186 prophage depends upon the phage function Tum, with the mutant phenotype of turbid plaques on mitomycin plates and the expression of which is controlled by the host LexA protein . Tum function, encoded near the right-hand end of the coliphage 186 chromosome, is under the control of promoter p95 . This promoter is overlapped by a sequence closely related to the consensus sequence of the LexA-binding site . It is proposed that inactivation of LexA after UV irradiation (or by genetic means) leads to prophage induction by permitting expression of Tum which, by unknown means, induces prophage . This mechanism is basically different from that seen with the UV-inducible lambdoid coliphages, which are not regulated by LexA. Proc Natl Acad Sci U S A, 1989 Jul, 86(14), 5320 - 4 Escherichia coli SecB protein associates with exported protein precursors in vivo; Kumamoto CA; The product of the Escherichia coli secB gene is required for efficient export of proteins across the cytoplasmic membrane . The studies described in this report show that in wild-type growing cells, SecB protein associates with precursor forms of exported proteins, such as the periplasmic maltose-binding protein (MBP) and the outer-membrane proteins LamB and OmpA . In contrast, the cytoplasmic protein beta-galactosidase was not found in association with SecB . Pulse-chase analysis showed that the SecB-precursor MBP complex was short lived, as expected for a complex that represents an intermediate in the protein-export pathway . The results support the hypothesis that SecB protein associates with exported protein precursors in the cytoplasm and dissociates prior to or during translocation of precursors across the cell membrane. Proc Natl Acad Sci U S A, 1989 Jul, 86(14), 5232 - 6 Labeling the peptidyltransferase center of the Escherichia coli ribosome with photoreactive tRNA(Phe) derivatives containing azidoadenosine at the 3' end of the acceptor arm: a model of the tRNA-ribosome complex; Wower J et al.; Photoreactive derivatives of yeast tRNA(Phe) containing 2-azidoadenosine (2N3A) at position 73 or 76 have been crosslinked to the peptidyl site of Escherichia coli ribosomes . Covalent tRNA-ribosome attachment was dependent upon the replacement of adenosine by 2N3A in the tRNA, irradiation with 300-nm light, and the presence of poly(U) . In all cases, the modified tRNAs became crosslinked exclusively to 50S ribosomal subunits . While the tRNA derivative containing 2N3A at position 73 labeled only protein L27, that containing 2N3A at position 76 labeled proteins L15, L16, and L27 as well as a segment of the 23S rRNA . The site of crosslinking in the rRNA was identified as guanosine-1945, which lies within a highly conserved sequence adjacent to a number of modified bases and has not until now been identified at the peptidyltransferase center . On the basis of these results, and previously reported crosslinks from tRNA containing 8-azidoadenosine in the 3'-terminal -A-C-C-A sequence {Wower, J., Hixson, S . S . & Zimmermann, R . A . (1988) Biochemistry 27, 8114-8121}, we propose a model for the arrangement of tRNA molecules at the peptidyl and aminoacyl sites that is consistent with most of the information available about the location of the peptidyltransferase center and the decoding domain of the E . coli ribosome. Proc Natl Acad Sci U S A, 1989 Jul, 86(14), 5222 - 6 Physical association of the 2,6-diamino-4-hydroxy-5N-formamidopyrimidine-DNA glycosylase of Escherichia coli and an activity nicking DNA at apurinic/apyrimidinic sites; O'Connor TR et al.; The 2,6-diamino-4-hydroxy-5N-formamidopyrimidine (Fapy)-DNA glycosylase of Escherichia coli, which is coded for by the fpg gene, excises purine bases with ring-opened imidazoles . In addition to the DNA glycosylase activity, we report that the Fapy-DNA glycosylase of E . coli has an associated activity, resistant to EDTA, that nicks DNA at apurinic/apyrimidinic (AP) sites . The levels of Fapy-DNA glycosylase and AP-nicking activity were parallel in crude lysates of E . coli HB101 harboring different plasmids constructed from the fpg gene . The fpg gene is different from the xth, nth, and nfo genes of E . coli, whose gene products also cleave DNA at AP sites . The Fapy-DNA glycosylase was purified to electrophoretic homogeneity . During this purification, the Fapy-DNA glycosylase copurified with an AP-nicking activity using chromatographic separations based on ion-exchange, molecular weight exclusion, and hydrophobicity . The cleavage at AP sites by the Fapy-DNA glycosylase left a 5'-phosphomonoester nucleotide at one terminus . In addition, DNA containing reduced AP sites was not nicked by the Fapy-DNA glycosylase . These data suggest that the mechanism of cleavage involved beta elimination . Therefore, this activity of the Fapy-DNA glycosylase nicking DNA at AP sites should be referred to as an AP lyase . The 3' terminus did not prime nick-translation by E . coli DNA polymerase I . However, the 3' terminus becomes a substrate for nick-translation if first allowed to react with calf intestine phosphatase or the E . coli exonuclease III . These data suggest that the repair of the Fapy lesion at least to some extent results in the formation of both 5'- and 3'-phosphomonoester nucleotides and the release of the deoxyribose. Mutat Res, 1989 Jul, 226(3), 141 - 4 Non-mutability by ultraviolet light in uvrD recB derivatives of Escherichia coli WP2 uvrA is due to inhibition of RecA protein activation; Woodgate R et al.; The deficiency in UV mutagenesis in uvrD3 recB21 strains of E . coli is almost completely overcome by constitutive activation of RecA protein and expression of the SOS system (by recA730 or 43 degrees C treated recA441 lexA71) . When SOS was expressed but RecA protein not self-activated (recA441 lexA71 at 30 degrees C), uvrD3 recB21 still reduced UV mutagenesis at low doses . The uvrD3 recB21 combination is therefore inhibiting activation of RecA protein . It is suggested that the DNA unwinding activity of the products of the uvrD and recB genes may be involved in generating single-stranded DNA needed to activate RecA protein both for the cleavage of LexA repressor and for a further role in UV mutagenesis. J Ultrasound Med, 1989 Jul, 8(7), 361 - 5 Sonographic features of focal orchitis; Lentini JF et al.; High-resolution sonography is a very sensitive imaging modality for detecting intratesticular pathology and is an accurate means of distinguishing intratesticular lesions (usually malignant) from extratesticular ones (usually benign) . Unfortunately, there are no reliable sonographic criteria to distinguish testicular neoplasms from focal benign intratesticular lesions such as infarction, hemorrhage, or infection . We describe three cases of focal orchitis in which the sonographic features did allow a confident diagnosis of intratesticular infection . In each case a focal peripheral hypoechoic intratesticular abnormality was seen that was poorly defined or crescent-shaped, adjacent to an enlarged epididymis . The specific sonographic features suggest the diagnosis of focal orchitis and orchiectomy can be prevented . Rapid improvement (2 to 4 weeks) should be seen sonographically and in all cases the intratesticular lesions should be followed to complete resolution. J Trauma, 1989 Jul, 29(7), 1015 - 20 The immunosuppressive effects of the in vivo administration of endotoxin as influenced by macrophages; Ogle CK et al.; It is well documented that endotoxin can have immunosuppressive effects on lymphocytes and induce the production and secretion of monokines which act on the lymphocytes . To delineate the interaction between macrophages and lymphocytes more clearly, 0.15 mg of lipopolysaccharide (LPS) (E . coli 0111:B4) was injected into Hartley guinea pigs intraperitoneally twice a day for 7 days (saline for control group) . Seven days after the last injection, spleens were taken and lymphocyte proliferation was determined in the presence and absence of macrophages . When macrophages were present, there was a significant suppression of lymphocyte proliferation when PHA and PWM were used as mitogens . There was no suppression of proliferation when the macrophages were removed . Splenic macrophages were also cultured in the presence and absence of LPS and their supernatants analyzed for PGE2 and TXB2 . There was no significant difference between the endotoxin and control groups for PGE2 or TXB2 production in the presence and absence of LPS . However, the endotoxin group had significant decreases in serum levels of C3 postinjection of endotoxin which could indicate C3 degradation by LPS . Taken together these results give further evidence that macrophage products in addition to PGE2 can inhibit lymphocyte proliferation . C3 degradation products could possibly stimulate macrophages to produce inhibitors of lymphocyte proliferation or induce suppressor cells. FASEB J, 1989 Jul, 3(9), 2086 - 8 Similarity between tyrosyl-tRNA synthetase and the estrogen receptor; Baker ME; Residues 1-42 of Escherichia coli tyrosyl-tRNA synthetase are similar to residues 293-334 of the human estrogen receptor . A computer analysis yields a comparison score that is 8.2 standard deviations higher than that obtained with 10,000 comparisons of randomized sequences of these segments (P = 1.2 X 10(-16) . This part of tyrosyl-tRNA synthetase binds ATP, which suggests that residues 293-334 of the human estrogen receptor are part of an ATP binding site. Clin Chem, 1989 Jul, 35(7 Suppl), B7 - 12 Development of a vector system for the expression of bioengineered proteins; Snouwaert JN et al.; The low natural abundance of many proteins is a major factor in preventing their development as therapeutic or diagnostic tools . To circumvent this barrier, we have used synthetic oligonucleotide technology to construct a gene based on the sequence of a cDNA for human interleukin 6 (IL-6) . The synthetic gene encodes a cysteine-free, bioengineered rIL-6 protein, which is expressed at high concentrations in Escherichia coli as a tripartite fusion protein . Cleavage of the fusion protein with collagenase (EC 3.4.24.8) releases a 23-kDa rIL-6 protein that can be easily purified to homogeneity . This rIL-6 protein displays a range of biological activities similar to those of natural human IL-6, as demonstrated by its ability to (a) protect cells from viral infection and (b) stimulate the synthesis of fibrinogen in rat FAZA cells. J Mol Endocrinol, 1989 Jul, 3(1), 15 - 21 Expression of biologically active recombinant-derived chicken prolactin in Escherichia coli; Hanks MC et al.; The putative chicken prolactin (chPRL) cDNA clone PRL101 was manipulated in vitro and cloned into the Escherichia coli expression vector pKK2332 to produce a plasmid coding for recombinant-derived mature chPRL (R-chPRL) . Expression of this manipulated cDNA sequence in E . coli resulted in the production of a 23 kDa protein which cross-reacted with specific chPRL antisera in Western blots . The partially purified protein stimulated ring dove crop sac mucosa to proliferate in a PRL bioassay, demonstrating that the R-chPRL was biologically active . R-chPRL was expressed at a level of approximately 1.5% of total cell protein. Proc Natl Acad Sci U S A, 1989 Jul, 86(13), 4953 - 7 Export of an N-terminal fragment of Escherichia coli flagellin by a flagellum-specific pathway; Kuwajima G et al.; Flagellin and several other external components of the bacterial flagellum are thought to be exported, not by the general N-terminal signal peptide-dependent pathway, but by a flagellum-specific pathway involving a central channel in the flagellum itself . We have constructed a variety of mutant alleles of the Escherichia coli flagellin gene . Mutant flagellins with large internal deletions or truncations of their C-terminal region could still be exported, even though they could not assemble into filament . The most extreme example was a fragment containing only the N-terminal 183 residues of the 497-residue wild-type flagellin . This result suggests that the N-terminal region of flagellin contains a signal that enables the protein to be recognized and exported by the flagellum-specific pathway. Proc Natl Acad Sci U S A, 1989 Jul, 86(13), 4927 - 31 Mutation at position 791 in Escherichia coli 16S ribosomal RNA affects processes involved in the initiation of protein synthesis; Tapprich WE et al.; A single base was mutated from guanine to adenine at position 791 in 16S rRNA in the Escherichia coli rrnB operon on the multicopy plasmid pKK3535 . The plasmid-coded rRNA was processed and assembled into 30S ribosomal subunits in E . coli and caused a retardation of cell growth . The mutation affected crucial functional roles of the 30S subunit in the initiation of protein synthesis . The affinity of the mutant 30S subunits for 50S subunits was reduced and the association equilibrium constant for initiation factor 3 was decreased by a factor of 10 compared to wild-type 30S subunits . The interrelationship among the region of residue 790 in 16S rRNA, subunit association, and initiation factor 3 binding during initiation complex formation, as revealed by this study, offers insights into the functional role of rRNA in protein synthesis. Proc Natl Acad Sci U S A, 1989 Jul, 86(13), 4902 - 6 In vitro methylation of Escherichia coli 16S ribosomal RNA and 30S ribosomes; Negre D et al.; Treatment of synthetic 30S particles lacking all of the normally methylated nucleotides with S-adenosyl-{3H}methionine and either an S100 or ribosomal high salt wash extract resulted in ribosome-dependent incorporation of {3H}methyl groups into trichloroacetic acid-insoluble material . No incorporation was observed when naturally methylated isolated 30S particles were used, showing that methylation at unnatural sites did not occur . Enzymatic hydrolysis of the labeled RNA to nucleosides followed by HPLC analysis identified the {3H}methylated residues . Activities for the formation of N6-methyladenosine, N6-dimethyladenosine, 5-methylcytidine (m5C), 3-methyluridine, and N2-methylguanosine were found . Fractionation by ammonium sulfate partially resolved the different activities . All of the fractions with m5C activity were 6-8 times more active on synthetic unmethylated 16S RNA than on synthetic 30S ribosomes, whereas the N2-methylguanosine activity preferred 30S ribosomes to 16S RNA by a factor of more than 10 . The N6-methyladenosine and N6-dimethyladenosine activities were 30S ribosome-specific . The m5C activity present in the 55-85% ammonium sulfate fraction of the high salt wash yielded a maximum of 1.0 mol of m5C per mol of 16S RNA, although two m5C residues, positions 967 and 1407, are found in vivo . RNase protection by hybridization with the appropriate oligodeoxynucleotide identified the methylated residue as C-967 . Methylation of m5C-967 did not require prior methylation of G-966, and methylation of A-1518 and A-1519 was not dependent on prior methylation of G-1516. Proc Natl Acad Sci U S A, 1989 Jul, 86(13), 4872 - 6 Identification of critical regions in mouse granulocyte-macrophage colony-stimulating factor by scanning-deletion analysis; Shanafelt AB et al.; Structure-function relationships for mouse granulocyte-macrophage colony-stimulating factor were examined by generating a series of small deletions scanning the entire length of the molecule . Deletions of three amino acids were introduced at intervals of five amino acids by site-directed mutagenesis of the mature mouse granulocyte-macrophage colony-stimulating factor gene . The mutant proteins were expressed in Escherichia coli and assayed for biological activity . This procedure identified four regions critical to activity . These critical regions were further delineated by additional three-amino acid deletion mutants . Larger deletions at each terminus were also made, as well as changes of specific amino acid residues . The four critical regions span amino acid residues 18-22, 34-41, 52-61, and 94-115 . The disulfide bridge between Cys-51 and Cys-93 was also shown to be essential for activity, whereas that between Cys-85 and Cys-118 could be removed without loss of activity . The possible structural and/or functional roles of the critical regions are discussed. J Pediatr, 1989 Jul, 115(1), 40 - 5 Special virulence of the Escherichia coli O1:K1:H7 clone in acute pyelonephritis; Marild S et al.; The relationship between acute-phase responses and bacterial properties was studied in a population of 88 children with their first known episode of acute pyelonephritis . One strain from each patient was included in the study . Eighty-four of the patients were infected with Escherichia coli, which was assigned a clonotype according to the O:K:H stereotype; 55 patients carried one of the 12 multiply occurring clones . Globotetraosylceramide-specific (globo+) adhesion was present in 90% of these 12 clones, compared with 62% in the remaining 29 singly occurring clones . The patients infected with globo+ strains had significantly increased inflammatory reactions compared with patients with globo- strains . The O1:K1:H7 strain was the single most frequent clone (n = 14) that always expressed globo+ adhesins . Patients infected with O1:K1:H7 had an inflammatory response similar to that of other globo+ infections, but had a shorter duration of symptoms before diagnosis, higher fever, and higher peripheral leukocyte count . These results demonstrate special virulence of the O1:K1:H7 clone, reflected by the acuteness of onset of infection. J Bacteriol, 1989 Jul, 171(7), 4083 - 4 Use of the isocitrate dehydrogenase structural gene for attachment of e14 in Escherichia coli K-12; Hill CW et al.; The e14 element appears to be integrated into the Escherichia coli K-12 isocitrate dehydrogenase structural gene (icd) . In being integrated, it replaced the last 52 codons of the gene with a closely related sequence . The two versions of the icd gene produce proteins of the same length but differ by 12 base substitutions that would cause two conservative amino acid replacements. J Bacteriol, 1989 Jul, 171(7), 4009 - 18 Genetic analysis of transcriptional activation and repression in the Tn21 mer operon; Ross W et al.; Transcription of the Tn21 mercury resistance operon (mer) is controlled by the toxic metal cation Hg(II) . This control is mediated by the product of the merR gene, a 144-amino-acid protein which represses transcription of the structural genes (merTPCAD) in the absence of Hg(II) and activates transcription in the presence of Hg(II) . We have used a mer-lac transcriptional fusion to obtain regulatory mutants in this metal-responsive system . Some mutants were defective in Hg(II)-induced activation while retaining repression function (a- r+), others were defective in repression but not activation (a+ r-), and some had lost both functions (a- r-) . Mutations in three of the four cysteine residues of merR resulted in complete loss of Hg(II)-inducible activation but retention of the repressor function, suggesting that these residues serve as ligands for Hg(II) in the activation process . Other lesions adjacent to or very near these cysteines exhibited severely reduced activation and also retained repressor function . There were two putative helix-turn-helix (HTH) domains in merR, and mutants in each had very different phenotypes . A partially dominant mutation in the more amino-terminal region of the two putative HTH regions resulted in loss of both activation and repression (a- r-), consistent with a role for this region in DNA binding . Mutations in the more centrally located HTH region resulted only in loss of Hg(II)-induced activation (a- r+) . Lesions in the central and in the carboxy-terminal regions of merR exhibited both Hg(II)-independent and Hg(II)-dependent transcriptional activation, suggesting that elements important in the activation mechanism may be widely distributed in this relatively small protein . The sole cis-acting mutant obtained with this operon fusion strategy, a down-promoter mutation, lies in a highly conserved base in the -35 region of the merTPCAD promoter. J Bacteriol, 1989 Jul, 171(7), 3940 - 7 Cloning, sequencing, and mapping of the bacterioferritin gene (bfr) of Escherichia coli K-12; Andrews SC et al.; The bacterioferritin (BFR) of Escherichia coli K-12 is an iron-storage hemoprotein, previously identified as cytochrome b1 . The bacterioferritin gene (bfr) has been cloned, sequenced, and located in the E . coli linkage map . Initially a gene fusion encoding a BFR-lambda hybrid protein (Mr 21,000) was detected by immunoscreening a lambda gene bank containing Sau3A restriction fragments of E . coli DNA . The bfr gene was mapped to 73 min (the str-spc region) in the physical map of the E . coli chromosome by probing Southern blots of restriction digests of E . coli DNA with a fragment of the bfr gene . The intact bfr gene was then subcloned from the corresponding lambda phage from the gene library of Kohara et al . (Y . Kohara, K . Akiyama, and K . Isono, Cell 50:495-508, 1987) . The bfr gene comprises 474 base pairs and 158 amino acid codons (including the start codon), and it encodes a polypeptide having essentially the same size (Mr 18,495) and N-terminal sequence as the purified protein . A potential promoter sequence was detected in the 5' noncoding region, but it was not associated with an "iron box" sequence (i.e., a binding site for the iron-dependent Fur repressor protein) . BFR was amplified to 14% of the total protein in a bfr plasmid-containing strain . An additional unidentified gene (gen-64), encoding a relatively basic 64-residue polypeptide and having the same polarity as bfr, was detected upstream of the bfr gene. J Bacteriol, 1989 Jul, 171(7), 3689 - 95 Localization of the ribosome-releasing factor gene in the Escherichia coli chromosome; Ichikawa S et al.; The ribosome-releasing factor (RRF) gene was localized at a position between 2 and 6 min on the Escherichia coli chromosome by measuring the gene-dosage-dependent production of RRF in various E . coli F' merozygotes . This position was confirmed and refined by using a nucleotide probe corresponding to a 16-amino-acid sequence in RRF . It was found that the RRF gene was contained in pLC 6-32 of the Clark-Carbon Gene Bank . Restriction enzyme mapping of E . coli genomic DNA with the above probe led us to conclude that the RRF gene is situated in the 4-min region, somewhere downstream (clockwise) of the elongation factor Ts gene, tsf . A pLC 6-32-derived DNA fragment which carries the RRF gene was found to contain a partial sequence of tsf . The exact location of the translational initiation site of the RRF gene was determined to be 1.1 kilobases downstream from the translational termination site of tsf . The RRF gene is designated frr. J Bacteriol, 1989 Jul, 171(7), 3619 - 28 Isolation and characterization of isoprene mutants of Escherichia coli; Sherman MM et al.; Isoprenoid compounds are found in all organisms . In Escherichia coli the isoprene pathway has three distinct branches: the modification of tRNA; the respiratory quinones ubiquinone and menaquinone; and the dolichols, which are long-chain alcohols involved in cell wall biosynthesis . Very little is known about procaryotic isoprene biosynthesis compared with what is known about eucaryote isoprene biosynthesis . This study approached some of the questions about isoprenoid biosynthesis and regulation in procaryotes by isolating and characterizing mutants in E . coli . Mutants were selected by determining their resistance to low levels of aminoglycoside antibiotics, which require an electron transport chain for uptake into bacterial cells . The mutants were characterized with regard to their phenotypes, map positions, enzymatic activities, and total ubiquinone content . In particular, the enzymes studied were isopentenyldiphosphate delta-isomerase (EC 5.3.3.2), farnesyldiphosphate synthetase (EC 2.5.1.1), and higher prenyl transferases. J Bacteriol, 1989 Jul, 171(7), 3609 - 18 Control of transducer methylation levels in Escherichia coli: investigation of components essential for modulation of methylation and demethylation reactions; Russell CB et al.; During bacterial chemotaxis in Escherichia coli, adaptation is accomplished by reversible methylation of the transmembrane signal transducers . Methyl groups are added by the CheR protein in a slow response to attractants and removed by the CheB protein in response to repellents . The methylesterase activity of the CheB protein is modulated by a factor that is controlled in a global fashion throughout the cell . By controlling the level of expression of the cheR, cheB, and transducer genes with exogenous promoters on multicopy plasmids, we demonstrate that the modulating factor exists in stoichiometric concentrations relative to CheB protein and that the generation or efficacy of this factor requires the cheA and/or cheW gene products, suggesting that phosphorylation of the methylesterase by CheA may be involved in its global activation . We show that in the absence of any modulation of the CheB activity, the CheR methyltransferase activity is modulated in a local fashion at the transducers, most likely as a result of a conformational change in the transducer protein brought about by the binding of ligand, and does not require CheA or CheW. J Bacteriol, 1989 Jul, 171(7), 3591 - 6 Inhibition of protein synthesis transiently stimulates initiation of minichromosome replication in Escherichia coli; Weinberger M et al.; Replication of oriC-dependent minichromosomes was found to be transiently stimulated when protein synthesis was inhibited by the addition of chloramphenicol . Initiation of replication was also induced by amino acid starvation of relA mutant strains and a nutritional upshift . The results are explained on the basis that these treatments rendered RNA polymerase more available for participation in the initiation process . As a consequence, the oriC duplex may be transcriptionally activated to an open form, a necessary prerequisite for DNA polymerization. Infect Immun, 1989 Jul, 57(7), 1928 - 35 Invasive potential of noncytotoxic enteropathogenic Escherichia coli in an in vitro Henle 407 cell model; Miliotis MD et al.; The invasive capacity of 13 enteropathogenic Escherichia coli strains was assessed in vitro in Henle 407 cell culture . Both fluorescent microscopy of infected monolayers stained with acridine orange and electron microscopy revealed the presence of intracellular bacteria . As shown by acridine orange-stained infected monolayers, the number of internalized bacteria increased with time . Monolayers infected for 3 h were treated with antibiotics and either {14C}glutamine or {3H}leucine and incubated for various time intervals, after which the amount of radioactivity present in the washed monolayers was measured . A significant (P less than 0.005) increase in uptake was evident for up to 4 h after the addition of radiolabeled amino acid . This finding was confirmed by an increase in bacterial number in cultured cells and in protein concentration of infected cells with time . None of the South African enteropathogenic E . coli isolates used in these studies produced Vero cytotoxin . These findings demonstrate that, in addition to adherence, cell penetration and intracellular multiplication take place in epithelial cell-derived tissue culture cells infected by enteropathogenic E . coli. Dev Biol, 1989 Jul, 134(1), 85 - 102 Developmental changes in phosphorylation state of neurofilament proteins in the chick embryonic optic nerve; Go MJ et al.; Developmental changes in the phosphorylation state of neurofilament proteins (NFPs) in the chick embryonic optic nerve were histochemically and biochemically studied using monoclonal antibody (MAb) 82E10 specific to the highly phosphorylated components of high (180K)- and middle (160K)-molecular-weight subunits of neurofilament (NF) in the chicken . Cross sections of developing embryonic optic nerve were studied by enzyme immunohistochemistry using this MAb . The staining pattern showed marked changes with the developmental stage . In 6-day embryos (E6) the entire cross section was stained, whereas in E10 only about a ventroposterior half of the cross section was stained . In E14 nearly the entire area of the cross section became unstained . Thereafter, the immunoreactivity reappeared and gradually increased, such that in E20 the entire cross section became immunopositive again . Electrophoretic and immunoblot analyses were made on optic nerves dissected out of embryos of various stages . The 82E10 immunoreactivity at the position of NF-M underwent a transient loss in E14 in parallel with the time course of histochemical change . Two-dimensional gels stained for protein further showed that the highly phosphorylated form of NF-M is transiently lost from embryonic optic nerve in E14, while the less phosphorylated form persists throughout the embryonic developmental stages . In order to understand the orderly loss of the 82E10 immunoreactivity in relation to retinotopic and chronotopic organizations of the fibers in the embryonic optic nerve, retinal injection of a fluorescent dye DiI as an anterograde tracing marker for selected fibers was utilized . An ordered arrangement of the fibers was present within the embryonic optic pathway, suggesting that the orderly loss of the 82E10 immunoreactivity in the embryonic optic nerve reflects the chronological order of the optic axons . These changes in the phosphorylation state of NFPs in the embryonic optic nerve presumably reflect dynamic changes of the neuronal cytoskeleton at certain stages during development. Mol Biol (Mosk), 1989 Jul-Aug, 23(4), 1113 - 23 {ID-similar sequences in various types of DNA clones}; Korotkov EV et al.; The sequence of rat ID repeat was compared with those of Escherichia coli, Drosophila melanogaster, chicken, rat, mouse and human clones and clones with t-RNA gene from EMBL-5 date bank . This comparison was made bearing on the determination of the level of mutual information between the sequences compared . The non-canonical similarity of the ID repeat sequence with the tRNA genes was found . It is revealed by the conservation of purine or pyrimidine sites in the sequences compared . In human and mouse clones purine-pyrimidine copies of rat ID sequence were also found . In some cases these sequences were flanked by short direct repeats, they contained also poly(A)-like sequences . The possible functional meaning and evolutional origin of the revealed relations are discussed. Mol Biol (Mosk), 1989 Jul-Aug, 23(4), 1013 - 21 {Structuro-functional organization of segments of Co1A plasmid DNA, determining its stable inheritance}; Kolot MN et al.; Two par regions were localized within the structure of a small colicinogenic plasmid ColA . One of them functions at the expense of plasmid multimere resolution . Analysis of the nucleotide sequence of the region revealed the existence of essential homology with the par locus of plasmid ColE1 . As compared to E . coli C600, the function of multimere forms' resolution of plasmid DNA in E . coli C is reduced or absent due to par regions of the ColE1 type . Par regions of various degrees of homology with the par locus of ColE1 were localized by Southern hybridization within the structure of colicinogenic plasmids ColN and ColD . The stabilization of the colicinogenic plasmids is believed to be also determined by the functioning of genes connected with the synthesis and action of colicin. Mol Microbiol, 1989 Jul, 3(7), 925 - 31 Regulation of the phase switch controlling expression of type 1 fimbriae in Escherichia coli; Pallesen L et al.; The expression of Escherichia coli type 1 fimbriae is phase-variable i.e . the bacterial cell is either fimbriated or non-fimbriated . The transition from one state to the other is caused by the change in configuration of an invertible DNA segment harbouring the promoter of the fimA gene . The position of this phase switch is controlled by two proteins, FimB and FimE, which mediate an 'on' or 'off' configuration of the switch, respectively . In this study, we have investigated how these proteins control the switch by means of fim-lac fusions on low-copy-number plasmids . It was found, by in trans and cis complementation, that the ratio of fimB to fimE and the total concentration of the gene products determine the configuration of the switch as well as the frequency of phase switching . It was also shown that transcription occurs from the promoter located at the phase switch when this is in the 'off' configuration . This suggests a regulatory mechanism, since the resulting transcript would be anti-sense to the fimE transcript. Acta Trop, 1989 Jul, 46(4), 229 - 38 Assessment of the prevalence and titer of antibodies to a candidate schistosomiasis vaccine molecule, Sj26, in several human serum banks; Lightowlers MW et al.; Using immunoassay and immunoblotting approaches, antibodies to Sj26, a glutathione S-transferase molecule (Mr = 26,000) of Schistosoma japonicum worms that is a vaccine candidate, have been sought in three large human serum banks . For these studies, a near-native recombinant Sj26 molecule produced in Escherichia coli was used, generally in ELISAs . Anti-Sj26 activity was detected readily in a high proportion of the sera at titres below 1:400 and appeared to be largely protein A-binding IgG antibodies . No differences in the prevalence of anti-Sj26 antibodies were noted in sera from entirely normal individuals or those with a variety of parasitic infections, but never exposed to S . japonicum . The stimulus responsible for induction of these low titre, probably low affinity antibodies in humans remains unknown. Biochem Int, 1989 Jul, 19(1), 173 - 83 Effect of heat-stable and heat-labile enterotoxins of Escherichia coli on intestinal brush border membrane enzymes of mice; Kohli M et al.; The activities of intestinal brush border membrane (BBM) enzymes alkaline phosphatase, maltase, lactase, sucrase, gamma-glutamyl transpeptidase and leucine aminopeptidase were determined in intestinal homogenates and purified BBMs from control, heat-stable and heat-labile enterotoxin treated mice . The activities of all the enzymes except lactase were decreased significantly (p less than 0.01) in homogenates while increased significantly (p less than 0.001) in BBMs of experimental groups as compared to controls . Calmodulin activities were increased significantly (p less than 0.01) as compared to control in heat-stable enterotoxin treated mice but remained unaltered in heat-labile enterotoxin treated mice . DNA contents of intestinal homogenates were decreased in experimental groups demonstrating the decrease in cell number in these groups . The altered BBM enzyme activities could not be attributed to changes in calmodulin activities . The increase in enzyme activities in BBMs may reflect a compensatory phenomenon in the remaining cells. Immunology, 1989 Jul, 67(3), 401 - 7 Role of cloned virulence factors (mannose-resistant haemagglutination, mannose-resistant adhesions) from uropathogenic Escherichia coli strains in the release of inflammatory mediators from neutrophils and mast cells; Konig W et al.; Genetically cloned E . coli strains expressing cloned virulence factors were studied with regard to their capability to induce inflammatory mediator release from various target cells . Among the strains were E . coli strains with mannose-resistant haemagglutination (MRH+) and mannose-resistant adhesins, e.g . E . coli 536/21 pANN 801/4, E . coli 536/21 pANN 921 and E . coli 536/21 pANN 801-1 . In comparison, E . coli 536/21, E . coli 536/21 pGB 30 int and E . coli K12, without and with mannose-sensitive haemagglutination (MSH +/-), and adhesins were studied . The properties of the various strains for human PMN with regard to adherence and phagocytosis, chemiluminescence, 5-lipoxygenase activation of arachidonic acid, leukotriene formation, granular enzyme release and release of histamine from rat mast cells were analysed . It is evident that the various biochemical processes of cell activation are dissociated events . The highest chemiluminescence response is obtained with strains expressing MSH+, P-MRH+ or S-MRH+; the presence of S-adhesins suppressed the response . Highest leukotriene formation is obtained with E . coli 536/21 pANN 801-4, while E . coli with MSH was inactive . The concomitant presence of haemolysin secretion enhanced mediator release significantly . Our data suggest a potent role for mannose-resistant haemagglutination (MRH), adhesins and haemolysin as virulence factors in inducing the release of inflammatory mediators. J Infect Dis, 1989 Jul, 160(1), 136 - 41 Production of diarrhea in the rabbit by a mutant of Escherichia coli (RDEC-1) that does not express adherence (AF/R1) pili; Cantey JR et al.; Escherichia coli (RDEC-1) adheres to Peyer's patch and absorptive epithelium in the rabbit in the closely adhering manner characteristic of enteropathogenic and enterohemorrhagic E . coli . An adherence pilus (AF/R1) is important for adherence to Peyer's patch M cells in vivo and ileal brush borders in vitro . A nonpiliated mutant (42-2-37-8) of the RDEC-1 strain colonized the gut lumen less readily than the parent strain . The mutant adhered infrequently to Peyer's patch lymphoid follicle epithelium (22% vs . 84%, P less than .0001) . Ileal close adherence was less frequent at 3 d, but by 9 d after inoculation had increased, approaching that of the parent RDEC-1 strain . However, adherence was focal, and fewer bacteria were present at each adherence site compared with the parent RDEC-1 strain . The result was a lower frequency of diarrhea (34% vs . 65%, P = .003) and mortality (9.4% vs . 27%, P = .035) with the 42-2-37-8 strain . Loss of AF/R1 pili compromised the ability of the RDEC-1 strain to adhere to Peyer's patch and absorptive epithelium and to produce diarrhea. J Infect Dis, 1989 Jul, 160(1), 131 - 5 Serotype-specific prevalence of Escherichia coli strains with EPEC adherence factor genes in infants with and without diarrhea in São Paulo, Brazil; Gomes TA et al.; To examine interrelationships of classic enteropathogenic Escherichia coli (EPEC) serotypes, EPEC adherence factor (EAF) genes, and diarrheal disease, E . coli were studied from stools of 500 infants less than 1 y of age with acute diarrhea and 500 age-matched controls . EAF-containing (EAF+) E . coli of three common classic EPEC serotypes (O111:H-, odds ratio {OR} 36.0; O111:H2, OR 55.0; O119:H6, OR 3.7) were individually strongly associated with diarrhea, as were EAF+ strains of less common classic serotypes combined (OR 5.3) . Among EPEC serogroups, neither EAF+ strains of nonclassic serotypes (OR 1.8) nor EAF-strains of classic (OR 2.2) or nonclassic (OR 1.4) serotypes were significantly associated with diarrhea . At least one EAF+ non-EPEC serogroup serotype (O88:H25) may represent an unrecognized EPEC serotype . Serotype-specific variation in the association of EAF+ E . coli with diarrhea suggests that other factors are also important in determining virulence; thus, both EAF detection and E . coli serotyping are desirable in studying the etiology of diarrheal disease. Infect Immun, 1989 Jul, 57(7), 2256 - 9 Binding of Escherichia coli S fimbriae to cultured human endothelial cells; Parkkinen J et al.; The binding of Escherichia coli adhesins to human umbilical vein endothelial cells was studied by a cell monolayer enzyme-linked immunosorbent assay . S fimbriae displayed a concentration-dependent and saturable binding to the endothelial cells which was mediated by their sialylgalactoside-specific lectin activity . P fimbriae exhibited only low binding, and type 1 fimbriae exhibited no binding to these cells. Plant Mol Biol, 1989 Jul, 13(1), 69 - 79 Cloning and expression of the chloroplast-encoded rbcL and rbcS genes from the marine diatom Cylindrotheca sp . strain N1; Hwang SR et al.; Both the rbcL and rbcS genes, encoding the large and small subunits, respectively, of ribulose 1,5-bisphosphate carboxylase/oxygenase, have been found to be encoded by chloroplast DNA in the marine diatom Cylindrotheca sp . N1 . The rbcS gene in this diatom was found to be adjacent to the rbcL gene by a combination of: (i) Southern-blotting analyses, using heterologous probes; (ii) examination of recombinant proteins synthesized in Escherichia coli, directed by cloned rbcL/rbcS genes; and (iii) synthesis of enzymatically active heterologous Rubisco protein in vivo by recombinant DNA procedures using large subunits of Anacystis nidulans and small subunits of Cylindrotheca sp . N1 . It appears that two copies of rbcL and rbcS genes are encoded by the chloroplast DNA of this diatom. J Gen Microbiol, 1989 Jul, 135 ( Pt 7), 1865 - 74 Mutations affecting the cytochrome d-containing oxidase complex of Escherichia coli K12: identification and mapping of a fourth locus, cydD; Poole RK et al.; A mutant of Escherichia coli K12 has been isolated affected in a gene, designated cydD, distinct from the three previously described loci involved in the synthesis of assembly of the cytochrome bd oxidase complex . The mutant, obtained by nitrosoguanidine mutagenesis, lacks the spectroscopically detectable components of this oxidase, namely cytochromes b558, b595 and d . Cytochrome oxidase o is the sole CO-binding cytochrome in membranes of the mutant, but the soluble haemoprotein b-590 and catalase activity appear unaffected . Discrimination between Cyd+ and Cyd- strains is facilitated by the development of a defined low-phosphate medium that allows the inclusion of Zn2+ as well as azide, inhibitors of respiratory electron transfer particularly via cytochrome o . Mapping with F-prime factors and by P1 cotransductional frequencies shows the mutation to map near 19.3 min on the E . coli chromosome, distinct from cydC, which maps at 18.9 min . The gene order in this region was tested in a three-factor cross and demonstrates the order zbj::Tn10(YYC199)-cydD-aroA, consistent with cotransduction frequencies. ASAIO Trans, 1989 Jul-Sep, 35(3), 341 - 3 Novel mechanical assistance in the treatment of endotoxic and septicemic shock; Hanasawa K et al.; Systemic sepsis is a frequent and fatal complication of postoperative patients . More recently, therapeutic trials of plasma or blood exchange are performed in septic patients for the purpose of reducing both endotoxins and albumin-bound toxins . As an alternate approach such as this for the removal of endotoxin directly from the blood, the authors recently developed polymyxin B immobilized fiber (PMX-F) as a biomaterial for selectively detoxifying endotoxin . That this newly invented PMX-F neutralizes a sufficient amount of endotoxin in vitro was reported previously . In ex vivo experiments, direct hemoperfusion by PMX-F was performed on purified endotoxin injected canine and on live Escherichia coli induced septic dogs . Only 5% (1/20) survived in the control group, but 75% (30/40) survived in the treated group . Septic dogs died within 18 hours after bacteria infusion in the control group . But all in the treated group survived 3 days . Forty percent of them survived permanently . These observations indicate that PMX-F treatment, namely selective removal of endotoxin in the endotoxic and septicaemic dogs prolongs or increases the chances of survival . Blood compatibility of PMX-F was also evaluated both in vitro and in vivo . With platelets it was shown to be fairly good and with red blood cells, white blood cells, and proteins, extremely good . A preclinical study on the efficacy and safety of PMX-F throughout widespread experiments has just ended. Genetika, 1989 Jul, 25(7), 1160 - 7 {Participation of the uvrD gene in the precise excision of the Tn9 transposon}; Mukhamedshin EK et al.; HfeTn5-(04,06) and IfeTn9 mutations increase efficiency of precise excision of Tn5, Tn10 and decrease that of Tn9 . These mutations have been mapped in uvrD gene . In LFETn9 and UVRE502 mutants, the multicopy plasmid pEM61 carrying the cloned uvrD gene complements LFETn9- phenotype (Low Frequency Excision of Tn9) . These results indicate that the uvrD gene product plays different role in excision of transposons with long and short inverted repeats . The mechanism of this effect is discussed. Mol Microbiol, 1989 Jul, 3(7), 979 - 84 Specificity of insertion of IS91, an insertion sequence present in alpha-haemolysin plasmids of Escherichia coli; Mendiola MV et al.; We have determined the DNA sequences of eight different insertions of IS91 in a specifically engineered recipient plasmid of known DNA sequence (pSU300) . The sequences at the termini of IS91 are 5'-CGAGTAGG...CCTATCGAT . IS91 inserts specifically 5' to either one of the tetranucleotides 5'-GAAC or 5'-CAAG, and always in the same relative orientation with respect to the sequence of the target . Except in one special case, no duplications of the recipient DNA were produced at the site of insertion. EMBO J, 1989 Jul, 8(7), 2101 - 9 Mutational analysis of IS10's outside end; Huisman O et al.; We present the genetic analysis of a large number of mutations in the outside end of insertion sequence IS10 . (i) The terminal inverted repeat sequence is probably the primary site of transposase binding . Mutations in this region fall into phenotypic classes which correspond to their map locations, suggesting that this region may consist of several distinct functional segments . Similarities between the organization of IS10's inverted repeat and those of other transposable elements are discussed . (ii) Base pairs 23-42 include a consensus binding sequence for one of the IS10 transposition host factors, IHF . The phenotypes of mutations in this region suggest that IHF is the major host factor for outside-end transposition activity in vivo and that base pairs throughout this region are important for the IHF interaction . (iii) Mutations in bp 43-61 do not affect outside-end transposition activity but do affect, in expected ways, previously identified determinants involved in expression and regulation of transposase . (iv) Some mutations in bp 23-42 also affect transposase expression; the possibility that IHF negatively regulates transcription initiation is discussed. Biochem Cell Biol, 1989 Jul, 67(7), 327 - 31 Investigation of adenylate kinase from Escherichia coli and its interaction with nucleotides by Fourier transform infrared spectroscopy; Arrondo JL et al.; The secondary structure of adenylate kinase (EC 2.7.4.3) from E . coli was investigated under various conditions using Fourier transform infrared spectroscopy . The overall band contour of the conformation-sensitive amide I mode indicates that in HEPES buffer (pH 7.4) the major structure of the protein is alpha-helical . A more detailed estimate obtained from decomposition of the amide I band into its constituent component bands gives 50% alpha-helix, 26% beta-structure, 15% turns and loops, and about 9% nonrepetitive unordered structures . Binding of nucleotide (e.g., ATP) to the donor site decreases the beta-content and shifts the amide I band to higher wavenumbers, whereas binding of nucleotide (e.g., AMP) to the acceptor site does not produce any change in conformation of the protein . These results agree with the protection by ATP and lack of protection by AMP when adenylate kinase is digested with trypsin . The effect of protein denaturing agents and conditions (temperature, high pH, sodium dodecyl sulfate) on changes in the protein conformation as revealed by the conformation-sensitive amide I bands is discussed. Plasmid, 1989 Jul, 22(1), 59 - 69 Cloning and genetic analysis of tra cistrons of the Tra 2/Tra 3 region of plasmid RP1; Palombo EA et al.; Transfer-defective mutants of the 10.4-kb Tra 2/Tra 3 region of RP1 were identified by their ability to be complemented by clones carrying all or part of this region . The respective mutations occurred in six cistrons whose order (traA, B, E, R, P, Q) and location were determined by deletion and insertion mapping . The cistrons occupy a minimum of 5.5 kb with the most distal, traA, spanning the 28.0-kb map position and traR the KpnI site at map position 24.1 kb . Each cistron is expressed independently, as Tn5 or Tn504 insertions in any one cistron do not affect the other five . The phenotypes controlled by each cistron suggest that all contribute to pilus biosynthesis/function while three (traB, R, and P) also contribute to surface exclusion . Given the occurrence of tra cistrons in the "silent" region between Tra 2 and Tra 3 we propose that the epithet "Tra 2" should be used to describe this entire region. Plasmid, 1989 Jul, 22(1), 1 - 9 Intermolecular plasmid recombination in fibroblasts from humans with DNA damage-processing defects; Timme TL et al.; We have evaluated the ability of immortalized human fibroblasts to recombine transfected plasmid DNA . A number of cell lines from normal individuals and from patients with DNA damage-processing defects were examined . Two plasmid recombination substrates were derived from pSV2neo and contained nonoverlapping deletions in the aminoglycoside phosphotransferase II gene . Intermolecular recombination was assessed by two methods after cotransfection . In a short-term, extrachromosomal recombination assay, low molecular weight DNA was extracted from the human cells 48 h after transfection, and recombinant plasmids were detected by transformation into appropriate indicator bacteria . In a long-term stable recombination assay the fibroblasts were cotransfected and G418-resistant colonies allowed to form . By the former assay all but two cultures were recombination-proficient, whereas all were recombination-proficient by the latter assay . The efficiency of transfection of human cells with plasmids appears to be a major variable affecting recombination . Recombination can be stimulated by uv irradiation of plasmid DNA prior to transfection . Cells from patients with Fanconi anemia, ataxia telangiectasia, and xeroderma pigmentosum complementation groups A, C, D, E, and G are not defective at intermolecular plasmid recombination. Mol Gen Genet, 1989 Jul, 218(1), 50 - 6 Initiation of DNA replication in Escherichia coli after overproduction of the DnaA protein; Skarstad K et al.; Flow cytometry was used to study initiation of DNA replication in Escherichia coli K12 after induced expression of a plasmid-borne dnaA+ gene . When the dnaA gene was induced from either the plac or the lambda pL promoter initiation was stimulated, as evidenced by an increase in the number of origins and in DNA content per mass unit . During prolonged growth under inducing conditions the origin and DNA content per mass unit were stabilized at levels significantly higher than those found before induction or in similarly treated control cells . The largest increase was observed when using the stronger promoter lambda pL compared to plac . Synchrony of initiation was reasonably well maintained with elevated DnaA protein concentrations, indicating that simultaneous initiation of all origins was still preferred under these conditions . A reduced rate of replication fork movement was found in the presence of rifampin when the DnaA protein was overproduced . We conclude that increased synthesis levels or increased concentrations of the DnaA protein stimulate initiation of DNA replication . The data suggest that the DnaA protein may be the limiting factor for initiation under normal physiological conditions. Mol Gen Genet, 1989 Jul, 218(1), 18 - 24 The uvp1 gene of plasmid pR cooperates with mucAB genes in the DNA repair process; Gigliani F et al.; We show that a DNA fragment that contains the uvp1 gene of the plasmid pR directs the synthesis in Escherichia coli minicells of a protein of apparent molecular weight 20 kDa . Inspection of the nucleotide sequence of the region reveals an open reading frame that has the capacity to encode a protein of 198 amino acids . The uvp1 gene product has been found, in two different systems, to enhance the recombinational activity of E . coli cells . We have also observed a striking similarity to resolvase and invertase proteins . The significance of this finding for the function of the uvp1 gene product requires further investigation . We conclude that the uvp1 gene encodes a 20 kDa protein which appears to be responsible for enhancement of both UV survival and recombinational activity in E . coli. J Biochem (Tokyo), 1989 Jul, 106(1), 181 - 7 Protein sorting between the outer and inner mitochondrial membranes: submitochondrial localization of cytochrome c1 whose presequence is replaced by the amino-terminal region of a 70 kDa outer membrane protein; Nakai M et al.; The amino-terminal region of a 70 kDa mitochondrial outer membrane protein of yeast and the presequence of cytochrome c1, an inner membrane protein exposed to the intermembrane space, are thought to be responsible for localizing the proteins in their final destinations after synthesis in the cytosol . Gene fusion experiments were used to identify signals that are responsible for protein sorting between the outer and inner mitochondrial membranes . The submitochondrial localization of cytochrome c1 whose presequence was replaced by the amino-terminal region of the 70 kDa mitochondrial outer membrane protein has been investigated . We have also used an in vivo complementation assay to determine whether or not a 70k-cyt c1 fusion protein is functional . Both the first half and all of the presequence of cytochrome c1 can be replaced by the amino-terminal 12 or 29 residues of the 70 kDa protein for transport to the inner membrane and functional assembly into succinate-cytochrome c reductase . However, replacements by the amino-terminal 61 residues of the 70 kDa protein result in exclusive localization of the fusion proteins to the outer membrane, and the fusions cannot be assembled into the enzyme complex . These data indicate that a mitochondrial targeting signal alone is sufficient to direct cytochrome c1 of mature size to the inner membrane. Genetics, 1989 Jul, 122(3), 491 - 501 Identification and characterization of a new gene of Escherichia coli K-12 involved in outer membrane permeability; Sampson BA et al.; Using a genetic selection for mutations which allow large maltodextrins to cross the outer membrane of Escherichia coli in the absence of the LamB maltoporin, we have obtained and characterized two mutations that define a new locus of E . coli . We have designated this locus imp for increased membrane permeability . Mapping studies show that the imp gene resides at approximately 1.2 min on the E . coli chromosome . The mutations alter the permeability of the outer membrane resulting in increased sensitivity to detergents, antibiotics and dyes . The mutations are nonreverting and codominant . Genetic analysis of the mutations suggest that the imp gene is an essential gene . We describe a general cloning strategy that can be used to clone both dominant and recessive alleles . Using this technique, we have cloned the wild-type and mutant imp alleles onto a low copy number plasmid. Eur J Biochem, 1989 Jul 1, 182(3), 547 - 55 The expression and purification of human rhinovirus protease 3C; Knott JA et al.; Human rhinovirus type 14 protease 3C was expressed as a soluble and active protein in Escherichia coli . The protease was purified by a cationic-exchange step followed by gel filtration on a TSK 3000 column . The final yield of purified protease was in the range 0.5-1.0 mg/l culture grown to A550 = 1.0 . Sequence analysis revealed that greater than 90% of the N-terminal residues were methionine . The enzyme activity of the purified protease was measured by cleavage of a synthetic peptide representing a predicted Gln/Gly viral polyprotein cleavage site . A mutant protease (Cys146----Ser) was produced and purified in the same way . The yield of mutant protease 3C was approximately 150 micrograms/l from a culture grown to A550 = 1.0 . This mutant protease 3C did not cleave the synthetic peptide substrate. Proc Natl Acad Sci U S A, 1989 Jul, 86(14), 5237 - 41 The (A)BC excinuclease of Escherichia coli has only the UvrB and UvrC subunits in the incision complex; Orren DK et al.; The uvrA, uvrB, and uvrC genes control excision repair in Escherichia coli . Cells with mutations in any of these three genes cannot repair DNA by nucleotide excision . When the purified gene products--the UvrA, UvrB, and UvrC proteins--are mixed together, an excision nuclease is formed that incises on both sides of the damaged nucleotide in an ATP-dependent reaction; it has been presumed that the excision nuclease was an ABC complex containing all three Uvr proteins . To determine the stoichiometry of the subunits in the enzyme, we conducted hydrodynamic studies with mixtures of the subunits with or without DNA substrate . We found that without DNA the UvrA subunit is a dimer and that when UvrB protein is also present, a (UvrA)2(UvrB)1 complex forms . Without DNA no detectable interaction of either the UvrA or UvrB subunits or the (UvrA)2(UvrB)1 complex with the UvrC subunit occurs . Unexpectedly, with UV-irradiated DNA, the UvrA/UvrB ratio in isolated DNA-protein complexes is variable, and the ratio becomes infinitesimally low as the UvrA concentration in the reaction mixture decreases . Under conditions of saturating UvrB protein approximately one UvrB molecule binds to DNA per damaged site in a reaction that requires catalytic amounts of UvrA subunit . Addition of UvrC protein to purified UvrB-DNA complexes results in rapid incision of the DNA, presumably catalyzed by an excision nuclease containing only UvrB and UvrC subunits. Proc Natl Acad Sci U S A, 1989 Jul, 86(13), 5010 - 4 An apaH mutation causes AppppA to accumulate and affects motility and catabolite repression in Escherichia coli; Farr SB et al.; apaH- mutants lack the hydrolase responsible for degradation of AppppN dinucleotides in Escherichia coli and show a greater than or equal to 16-fold increase in AppppA under nonstress conditions . These mutants lack detectable activity of sigma F, a factor required for transcription of motility and chemotaxis genes . Expression of the flbB/flaI operon, thought to encode sigma F, is decreased in apaH- mutants, and there appears to be a general decrease in expression of genes regulated by cAMP-binding protein and cAMP as well. J Bacteriol, 1989 Jul, 171(7), 4071 - 2 Methionine aminopeptidase gene of Escherichia coli is essential for cell growth; Chang SY et al.; We localized the methionine aminopeptidase (map) gene on the Escherichia coli chromosome next to the rpsB gene at min 4 . Genetically modified strains with the chromosomal map gene under lac promoter control grew only in the presence of the lac operon inducer isopropyl-beta-thiogalactoside . Thus, methionine aminopeptidase is essential for cell growth. J Bacteriol, 1989 Jul, 171(7), 3996 - 4001 Effects of variation of inverted-repeat sequences on reactions mediated by the transposase of Tn21; Martin C et al.; The frequencies of one-ended transposition and normal transposition of derivatives of Tn21 that contain mutant inverted-repeat sequences (IRs) have been measured . In general, there was a linear relationship between the log of the frequency of one-ended transposition of a mutant IR and the log of the frequency of normal transposition of an element flanked by a wild-type IR at one end and by the mutant IR at the other . This implied that one-ended and normal transposition share the rate-limiting step that determines the frequency of transposition and that both IRs are involved in the rate-limiting step in normal transposition . Surprisingly, it was found that only the outer 18 base pairs of the IR of Tn21 engaged accurately in both one-ended and normal transposition, at about 1% of the frequency of the wild-type IR. J Bacteriol, 1989 Jul, 171(7), 3817 - 23 Oxygen, nitrate, and molybdenum regulation of dmsABC gene expression in Escherichia coli; Cotter PA et al.; Escherichia coli can respire anaerobically using either trimethylamine-N-oxide (TMAO) or dimethyl sulfoxide (DMSO) as the terminal electron acceptor for oxidative phosphorylation . To determine whether the regulation of the dmsABC genes, which encode a membrane-associated TMAO/DMSO reductase, are transcriptionally controlled in response to the availability of alternate electron acceptors, we constructed an operon fusion between the dmsA gene, along with its associated regulatory region, and lacZ+ . Expression of dmsA'-lacZ was stimulated 65-fold by anaerobiosis versus aerobiosis, while nitrate caused a 12-fold repression . Its expression, however, was unaffected by the presence of the alternate electron acceptors DMSO, TMAO, and fumarate . Anaerobic induction of dmsA'-lacZ was defective in an fnr mutant, thus establishing that Fnr is responsible for anaerobic activation of dmsABC . Repression of dmsA'-lacZ expression by nitrate was independent of oxygen and was shown to be mediated by the products of two genes, narL (frdR2) and narX . dmsA'-lacZ expression was also altered in chlD strains that are defective in molybdenum transport but not in chlA and chlE strains that are defective in molybdopterin cofactor biosynthesis, thus establishing that the molybdenum ion but not the ability to form a functional cofactor is required for regulation . Molybdenum was required both for complete induction of dmsA'-lacZ expression during anaerobic growth and for complete repression of dmsA'-lacZ by nitrate . Additionally, expression of dmsABC varied depending on the carbon source . Expression was highest when cells were grown on sorbitol. J Bacteriol, 1989 Jul, 171(7), 3810 - 6 Identification of a second gene involved in global regulation of fumarate reductase and other nitrate-controlled genes for anaerobic respiration in Escherichia coli; Kalman LV et al.; Fumarate reductase catalyzes the final step of anaerobic electron transport in Escherichia coli when fumarate is used as a terminal electron acceptor . Transcription of the fumarate reductase operon (frdABCD) was repressed when cells were grown in the presence of either of the preferred terminal electron acceptors, oxygen or nitrate, and was stimulated modestly by fumarate . We have previously identified a locus called frdR which pleiotropically affects nitrate repression of fumarate reductase, trimethylamine N-oxide reductase, and alcohol dehydrogenase gene expression and nitrate induction of nitrate reductase expression (L . V . Kalman and R . P . Gunsalus, J . Bacteriol . 170:623-629, 1988) . Transformation of various frdR mutants with plasmids identified two complementation groups, indicating that the frdR locus is composed of two genes . One class of mutants was not completely restored to wild-type frdA-lacZ expression or nitrate reductase induction when complemented with multicopy narX+ plasmids, whereas low-copy narX+ plasmid-containing strains were . A second class of frdR mutants was identified and shown to correspond to a previously described gene, narL (frdR2) . Complementation of these strains with multicopy narL+ plasmids resulted in superrepression of frdA-lacZ expression and moderate elevation of nitrate reductase expression . Multicopy plasmids containing both narL+ and narX+ or only narL+ were able to complement narL mutants, whereas narX+ plasmids complemented narX mutants only when present in a copy number approximately equal to that of narL . Both narL and narX mutants retained normal oxygen control of frdA-lacZ expression . Both types of mutants are pleiotropic, as evidenced by derepressed levels of the fumarate reductase and trimethylamine N-oxide reductase enzymes and by defective induction of nitrate reductase when cells were grown in the presence of nitrate . These results indicate that both the narL and narX gene products must be present in a defined ratio in the cell . We conclude that these proteins interact to effect normal nitrate control of the anaerobic electron transport-associated operons . From these studies, we propose that narX encodes a nitrate sensor protein while narL encodes a DNA-binding regulatory protein which together function in a manner analogous to other two-component regulatory systems. J Bacteriol, 1989 Jul, 171(7), 3803 - 9 Segregation of relaxed replicated dimers when DNA ligase and DNA polymerase I are limited during oriC-specific DNA replication; Munson BR et al.; An in vitro Escherichia coli oriC-specific DNA replication system was used to investigate the DNA replication pathways of oriC plasmids . When this system was perturbed by the DNA ligase inhibitor nicotinamide mononucleotide (NMN), alterations occurred in the initiation of DNA synthesis and processing of intermediates and DNA products . Addition of high concentrations of NMN soon after initiation resulted in the accumulation of open circular dimers (OC-OC) . These dimers were decatenated to open circular monomers (form II or OC), which were then processed to closed circular supercoiled monomers (form I or CC) products . After a delay, limited ligation of the interlinked dimers (OC-OC to CC-OC and CC-CC) also occurred . Similar results were obtained with replication protein extracts from polA mutants . The presence of NMN before any initiation events took place prolonged the existence of nicked template DNA and promoted, without a lag period, limited incorporation into form II molecules . This DNA synthesis was nonspecific with respect to oriC, as judged by DnaA protein dependence, and presumably occurred at nicks in the template DNA . These results are consistent with oriC-specific initiation requiring closed supercoiled molecules dependent on DNA ligase activity . The results also show that decatenation of dimers occurs readily on nicked dimer and represents an efficient pathway for processing replication intermediates in vitro. J Bacteriol, 1989 Jul, 171(7), 3760 - 6 DnaA protein overproduction abolishes cell cycle specificity of DNA replication from oriC in Escherichia coli; Pierucci O et al.; Initiation of DNA replication from oriC in Escherichia coli takes place at a specific time in the cell division cycle, whether the origin is located on a chromosome or a minichromosome, and requires participation of the product of the dnaA gene . The effects of overproduction of DnaA protein on the cell cycle specificity of the initiation event were determined by using minichromosome replication as the assay system . DnaA protein was overproduced by inducing the expression of plasmid-encoded dnaA genes under control of either the ptac or lambda pL promoter . Induction of DnaA protein synthesis caused a burst of minichromosome replication in cells at all ages in the division cycle . The magnitude of the burst was consistent with the initiation of one round of replication per minichromosome in all cells . The replication burst was followed by a period of reduced minichromosome replication, with the reduction being greater at 30 than at 41 degrees C . The results support the idea that the DnaA protein participates in oriC replication at a stage that is limiting for initiation . Excess DnaA protein enabled all cells to achieve the state required for initiation of DNA polymerization by either effecting or overriding the normal limiting process. J Bacteriol, 1989 Jul, 171(7), 3704 - 12 Multiple defects in Escherichia coli mutants lacking HU protein; Huisman O et al.; The HU protein isolated from Escherichia coli, composed of two partially homologous subunits, alpha and beta, shares some of the properties of eucaryotic histones and is a major constituent of the bacterial nucleoid . We report here the construction of double mutants totally lacking both subunits of HU protein . These mutants exhibited poor growth and a perturbation of cell division, resulting in the formation of anucleate cells . In the absence of HU, phage Mu was unable to grow, to lysogenize, or to carry out transposition. J Bacteriol, 1989 Jul, 171(7), 3641 - 9 Molecular analysis of the Escherichia coli recO gene; Morrison PT et al.; The plasmid pLC7-47, which contains lep, rnc, and era, was found to complement the UV-sensitive and recombination-deficient phenotypes caused by the recO1504::Tn5 mutation . Southern blotting analysis demonstrated that pLC7-47 contained a segment of Escherichia coli DNA that covered the region of the E . coli chromosome containing the recO1504::Tn5 mutation . A combination of deletion mapping and insertional mutagenesis localized the recO-complementing region to an approximately 1-kilobase region of a 1.6-kilobase BamHI fragment . The DNA sequence of the 1.6-kilobase BamHI fragment was determined and contained part of era and a 726-base-pair recO open reading frame . The recO open reading frame contained three possible translation start codons and could potentially encode a polypeptide of Mr 26,000 . Computer analysis indicated that the putative RecO protein had suboptimal codon usage and did not show significant homology with previously identified proteins whose sequences were present in protein data bases . A combination of primary sequence analysis and secondary structure predictions suggested that recO contains a mononucleotide-binding fold. Carcinogenesis, 1989 Jul, 10(7), 1299 - 306 Molecular mechanisms of alkylation sensitivity in Indian muntjac cell lines; Musk SR et al.; The responses of two Indian muntjac cell lines to two monofunctional alkylating agents were investigated . An SV40-transformed line (SVM) had an increased sensitivity to cell killing when compared to the other, euploid line (DM) after exposure both to methyl nitrosourea (MNU) and to dimethylsulphate (DMS) and also exhibited higher frequencies of sister chromatid exchanges (SCEs) following alkylation . The hypersensitivity of SVM to DMS correlates with the defective repair of single-strand breaks that results in the generation of long-lived breaks in the DNA following exposure, leading eventually to the formation of chromosome aberrations . In contrast no difference is seen in the formation of long-lived breaks in the DNA of SVM and DM after treatment with biologically relevant doses of MNU; in this case hypersensitivity may be due to the loss of O6-alkylguanine-DNA-alkyltransferase activity . The conclusion that the hypersensitivites of SVM to MNU and to DMS have different molecular bases is supported by transfection of SVM with plasmids containing the protein coding region of the Escherichia coli ada+ gene; subsequent expression within the cell corrects its hypersensitivity to the cytotoxic and SCE-inducing effects of MNU but has very little influence upon the lethality, SCE induction or the repair of long-lived DNA strand breaks after exposure to DMS. J Infect Dis, 1989 Jul, 160(1), 52 - 7 Polymyxin B prevents lipopolysaccharide-induced release of tumor necrosis factor-alpha from alveolar macrophages; Stokes DC et al.; Polymyxin B (PmB) blocks many of the toxic effects of lipopolysaccharide by mechanisms that are not yet understood . The production of tumor necrosis factor-alpha (TNF-alpha) by isolated rat alveolar macrophages in response to lipopolysaccharide and macrophage-activating factor was blocked by PmB at concentrations of 100, 10, and 1 micrograms/ml . Gentamicin enhanced rather than inhibited TNF production at the 100-micrograms/ml concentrations and had no effect at low concentration . Similar inhibitory effects were induced by PmB in an in vivo model in which rat macrophage TNF production was stimulated by intratracheally injected lipopolysaccharide . Because many of the effects of lipopolysaccharide are mediated by TNF, this inhibition provides a mechanism to explain the protection afforded by PmB against lipopolysaccharide-induced toxicity. Infect Immun, 1989 Jul, 57(7), 2223 - 9 Biological activities and chemical composition of purified tracheal cytotoxin of Bordetella pertussis; Cookson BT et al.; Specific destruction of ciliated epithelial cells lining the large airways is the primary respiratory tract cytopathology associated with human Bordetella pertussis infections . We have purified a single low-molecular-weight glycopeptide, tracheal cytotoxin (TCT), that appears to cause this pathology . By using a combination of solid-phase extraction and reversed-phase high-pressure liquid chromatography, about 700 nmol of biologically active peptide can be isolated from 1 liter of B . pertussis culture supernatant (approximately 60% yield) . TCT at concentrations of 1 microM destroyed the ciliated cell population when incubated with respiratory epithelium in vitro . This concentration of TCT is similar to the concentrations found in the culture supernatant of growing B . pertussis . Purified TCT also inhibited DNA synthesis of hamster trachea epithelial cells in a quantitative, dose-dependent fashion . Endotoxin was not detected in the purified material, and neither B . pertussis nor Escherichia coli endotoxin could duplicate the biological activities of TCT . Amino acid and amino sugar analyses of purified TCT revealed the presence of glucosamine, muramic acid, alanine, glutamic acid, and diaminopimelic acid in molar ratios of 1:1:2:1:1 . This suggests that TCT, the released ciliostatic principle of B . pertussis, is a disaccharide tetrapeptide subunit of peptidoglycan. J Virol, 1989 Jul, 63(7), 3016 - 25 Functional limits of oriP, the Epstein-Barr virus plasmid origin of replication; Chittenden T et al.; The Epstein-Barr virus (EBV) genome contains two cis-acting elements which are required for stable extrachromosomal plasmid maintenance in latently infected cells . The first consists of 20 30-base-pair (bp) repeats, each of which contains a DNA-binding site for EBV nuclear antigen 1 (EBNA-1), the trans-acting factor required for plasmid persistence . The second element is composed of a 65-bp dyad symmetry, containing four EBNA-1-binding sites . Deletion mutants were constructed which reduce the number of EBNA-1-binding sites in the 30-bp repeats, alter the number of EBNA-1-binding sites in the dyad region, or truncate the dyad element . The effect of the deletion mutations on plasmid maintenance was examined by transfecting recombinant plasmids, containing both the mutated EBV sequences and a drug resistance marker, into D98-Raji cells . The plasmids were tested for their ability to generate drug-resistant D98-Raji cell colonies and their capacity to be maintained in an extrachromosomal form without undergoing extensive rearrangements . EBV plasmids with 12 or 15 copies of the 30-bp repeats were wild type in both assays . Plasmids with just two or six copies of these repeated elements failed to generate d