Mol Gen Genet, 1984, 193(3), 473 - 8 Metabolic alterations mediated by 2-ketobutyrate in Escherichia coli K12; Danchin A et al.; We have previously proposed that 2-ketobutyrate is an alarmone in Escherichia coli . Circumstantial evidence suggested that the target of 2-ketobutyrate was the phosphoenol pyruvate: glycose phosphotransferase system (PTS) . We demonstrate here that the phosphorylated metabolites of the glycolytic pathway experience a dramatic downshift upon addition of 2-ketobutyrate (or its analogues) . In particular, fructose-1,6-diphosphate, glucose-6-phosphate, fructose-6-phosphate and acetyl-CoA concentrations drop by a factor of 10, 3, 4, and 5 respectively . This result is consistent with (i) an inhibition of the PTS by 2-ketobutyrate, (ii) a control of metabolism by fructose-1,6-diphosphate . Since fructose-1,6-diphosphate is an activator of phosphoenol pyruvate carboxylase and of pyruvate kinase, the concentration of their common substrate, phosphoenol pyruvate, does not decrease in parallel. J Gen Microbiol, 1984 Jan, 130 ( Pt 1), 83 - 8 An inducible phosphoenolpyruvate: dihydroxyacetone phosphotransferase system in Escherichia coli; Jin RZ et al.; A phosphoenolpyruvate: dihydroxyacetone phosphotransferase was induced in Escherichia coli grown on dihydroxyacetone as sole carbon source or in its presence . This is the first example of a triose which can be acted upon by the membrane complex to provide a central intermediate in glycolysis . The presence of this system explains the ability of a mutant, in which the ATP-dependent glycerol kinase is genetically replaced by a glycerol: NAD 2-oxidoreductase, to grow on glycerol. J Gen Microbiol, 1984 Jan, 130 ( Pt 1), 217 - 22 Degradation of Escherichia coli chromosomal and plasmid DNA in serum; Rozenberg-Arska M et al.; Incubation of serum-sensitive {3H}thymidine labelled Escherichia coli PC2166 (RSF1030) and E . coli AM1281 (pBR322) harbouring small plasmids (mol . wt 5.5 X 10(6) and 2.6 X 10(6} in serum resulted in killing of 99.9% of the bacteria within 15 min and in the release of 85% of the radioactivity into the medium after 1 h incubation . The fate of chromosomal and plasmid DNA during incubation of the bacteria in serum was analysed by measurement of the amount of DNA-associated radioactivity, by TCA precipitation, by agarose gel electrophoresis and by the capacity of DNA to transform competent acceptor bacteria . Chromosomal DNA and high molecular weight plasmid DNA were rapidly degraded after 1 h incubation of bacteria in serum . However, low molecular weight plasmid DNA was virtually unaffected and remained physicochemically as well as biologically intact during up to 4 h of incubation of bacteria in serum. J Gen Microbiol, 1984 Jan, 130 ( Pt 1), 209 - 15 Two new E colicins, E8 and E9, produced by a strain of Escherichia coli; Cooper PC et al.; We have isolated a strain of Escherichia coli from chicken caeca which produces two E colicins and colicin M . This strain has seven plasmids, five of which have been transferred to E . coli K12 . Two E . coli K12 derivatives which produce the two E colicins separately have been tested against seven standard E colicin producing strains which define seven different immunity groups . Our results indicate that these new E colicins define two further immunity groups, E8 and E9. EMBO J, 1984 Jan, 3(1), 91 - 4 Does codon composition influence ribosome function? Andersson SG, Buckingham RH, Kurland CG. Escherichia coli ribosomes pre-initiated with N-acetyl-Val-tRNAVal elongate strictly alternating poly(U-G) at a rate between eight and 12 peptide bonds per second per ribosome in vitro . Comparisons with poly(U)-primed poly(Phe) synthesis show that these systems function with the same rates which are close to those of protein synthesis in vivo . This indicates that, at least in vitro, codon composition has no marked influence on the speed of elongation when the concentration of ternary complex is saturating . Furthermore, the missense frequencies for the two polymers are within the same range: the missense substitution of Trp for Cys is 10(-4) and that of Met for Val is 10(-3) in the poly(U-G)-primed system . These data argue against models that explain the codon preference of certain gene families by postulating effects of high or low GC content of codons on the performance characteristics of ribosomes. Circ Shock, 1984, 12(2), 135 - 49 Continuous infusion of endotoxin from an osmotic pump in the conscious, unrestrained rat: a unique model of chronic endotoxemia; Fish RE et al.; Endotoxin (ET) was administered to conscious, unrestrained rats by continuous intravenous infusion from an Alzet osmotic pump . Delivery of ET was delayed 42 h after surgery by inserting a 100-cm coil of PE-60 tubing between pump and jugular vein . Rats were anorectic following onset of ET delivery; therefore control rats were either fed ad libitum or food-deprived (FD) to match the voluntary consumption of ET rats . Blood was collected from carotid catheters and oxygen consumption determined daily . Body weight, colon temperature, and plasma glucose were similar in ET and FD rats, but ET rats exhibited a transient hyperlactacidemia, progressive leukocytosis, and fall in hematocrit which was not seen in FD rats . Food deprivation resulted in a marked drop in plasma insulin which was not seen in ET rats, despite similar food intake and plasma glucose concentration . Oxygen consumption of ET rats was significantly greater than both fed and FD animals on days 1 and 2 of ET infusion, while mean arterial pressure and heart rate were similar to controls . A unique model of endotoxemia is presented which is characterized by a transient hypermetabolic state, and changes in plasma lactate and insulin levels, white cell count, and hematocrit, which cannot be attributed to food deprivation . The results suggest that ET may be important in the pathogenesis of hypermetabolic sepsis. Circ Shock, 1984, 12(1), 61 - 71 Effect of hemorrhagic shock on endotoxin-induced pulmonary hypertension and increased vascular permeability in unanesthetized sheep; Wong C et al.; The lung is very susceptible to sepsis or endotoxin injury in the trauma patient . We studied the effect of an episode of hemorrhagic shock and resuscitation on the prostaglandin-induced pulmonary hypertension and leukocyte-induced increased permeability phase of endotoxin lung injury . Eight unanesthetized sheep with chronic lung lymph fistula were bled 50% of blood volume for 2 hr, then resuscitated . Thromboxane, TxA2, levels increased from 0.1 to 0.6 ng/ml during shock, while blood white cell count decreased . Both parameters returned to baseline while lung lymph flow increased twofold during resuscitation with lymph being protein-poor, indicating no increase in permeability . Lung water was not increased but some pulmonary leukostasis was evident histologically after resuscitation . We then studied the effect of this process on all immediate endotoxin insult . Seven unanesthetized sheep were given 0.7 microgram/kg E . coli endotoxin alone, and again after shock and resuscitation, in paired studies performed 3 days apart . There was no difference in either the early pulmonary hypertension or the later increased permeability phase of endotoxin lung injury when comparing the paired studies, as measured by lymph flow and protein flux . Hemorrhagic shock, despite producing a transient increase in thromboxane and pulmonary leukocyte sequestration, does not accentuate the lung injury of endotoxin if the shock state is adequately resuscitated. Circ Shock, 1984, 12(1), 25 - 46 Effects of captopril on hemodynamic and metabolic parameters in awake endotoxemic Yucatan minipigs; Fettman MJ et al.; Angiotensin II (AII), a potent vasoconstrictor, contributes to ischemic and decompensatory phases of shock . Captopril may benefit vital organ and tissue perfusion by inhibiting AII formation . We fitted 13 approximately 50-kg pigs with jugular, portal, hepatic vein, and carotid artery catheters, and hepatic artery and portal vein flow cuffs to quantitate portosystemic and transhepatic kinetics . We placed them in slings 72 hr later and following a 3-hr control period, they were infused with E . coli endotoxin at 15 micrograms/kg/hr for 6 hr . We kept eight as controls and five received a primed (2 mg/kg) continuous infusion (2 mg/kg/hr) of captopril 1 hr after initiation of endotoxin . Arterial hypotension developed by 60 min and hypoglycemia by 100 min in both groups; captopril had no effect on these parameters . Blood lactate increased from 7.8-32.1 mg/dl 80 min postendotoxin, and for the third to fifth hours of endotoxin infusion was significantly higher than those of the control group . 6-3H-glucose-derived appearance (Ra) values remained at 1.88-2.35 mg/kg/min throughout the experiment . Glucose disappearance (Rd) values were elevated from 60-120 min of endotoxin, increasing to 2.68-3.13 mg/kg/min versus 1.86 mg/kg/min preendotoxin . These changes corresponded to those in lactate, and incurred only a brief significant net glucose deficit (%Rd-%Ra) that peaked at 61.1% at 100 min . For 140-360 min postendotoxin, glucose balance was at most 8.3% in deficit (200 min) and 13.6% in positive balance (280 min) versus a net deficit of up to 25.4% (220 min) for the untreated group for much of the experiment . Portal and hepatic venous blood flow in captopril-treated pigs was lower than that in untreated pigs before endotoxin (8.2 and 10.0 ml/kg/min versus 12.9 and 17.9 ml/kg/min, respectively), but was not depressed following endotoxin infusion, versus 50% reductions in the untreated pigs, postendotoxin . Captopril maintained hepatosplanchnic blood flow and effected modest improvements in glucose kinetics. Cancer Detect Prev, 1984, 7(1), 51 - 8 A pancreatic oncofetal antigen (POA): its characterization and application for enzyme immunoassay; Oguchi H et al.; We investigated the usefulness of enzyme immunoassay (EIA) for pancreatic oncofetal antigen (POA) . The crude POA isolated from POA-positive ascitic fluid of patients with pancreatic cancer was injected into rabbits to raise anti-POA serum . The adsorbed antiserum was used for EIA as anti-POA serum . For the establishment of EIA system for POA, anti-POA-Fab' fragment was conjugated to beta-D-galactosidase from Escherichia Coli . Normal subjects (205 controls) and 132 patients (47 with pancreatic cancer, 22 with chronic pancreatitis, and 63 with other malignant disease) were surveyed . The standard serum from patient M with pancreatic cancer was used in quantitatively determining serum POA levels; value was expressed arbitrarily as 1000U/ml . Normal upper limit of POA was defined as less than 400U/ml (mean + 2SD of normal subjects) . POA level higher than normal was observed in 72% of patients with pancreatic cancer, 23-44% of patients with other malignant diseases, and 18% of patients with chronic pancreatitis . The susceptibility of the isolated POA to several enzymes and chemical reagents was also studied . These results suggest the usefulness of EIA for POA in diagnosis of pancreatic cancer. Biokhimiia, 1984 Jan, 49(1), 160 - 2 {Cleavage of tRNA and rRNA at 7-methylguanine in the presence of methylated carrier RNA}; Zueva VS et al.; A method of site-specific cleavage of some tRNAs and rRNAs at the 7-methylguanine residue is described . After reduction of 7-methylguanine by sodium borohydride treatment in the presence of the exogenous carrier RNA methylated statistically by dimethylsulfate the polynucleotide chain is cut at the modified residue by aniline . It was shown that the previously used procedure which did not involve a methylated carrier RNA is not applicable for splitting rRNAs or low concentrations of tRNAs . The method described was successfully used for site-specific cleavage of 32P-terminally labelled yeast tRNAPhe and unlabelled E . coli 16S rRNA at 7-methylguanine position. Am Rev Respir Dis, 1984 Jan, 129(1), 72 - 5 Quantitation of leukocytes in bronchoalveolar lavage samples from rats after intravascular injection of endotoxin; Chang JC et al.; Although it has been demonstrated in several animal species that neutrophils aggregate in the pulmonary microvasculature after intravenous infusion of chemotactic factors or substances that activate the systemic complement system, studies in rabbits have revealed that affected neutrophils do not migrate out of capillaries into air spaces unless the infusion processes are combined with manipulative procedures involving the airways, such as intubation or instillation of an anesthetic agent . In this study, we report that after intravenous infusion of endotoxin (Escherichia coli) into Sprague-Dawley rats, in the absence of airway manipulation, the absolute number of neutrophils in bronchoalveolar lavage (BAL) samples increased significantly 24 and 48 h after injection . This represented nearly a 24-h delay from the time that maximal numbers of neutrophils were seen in lung tissue and approximately an 18-h delay from the time that maximal numbers appeared in peripheral blood . We also found that after infusion of endotoxin the number of pulmonary alveolar macrophages (PAM) recoverable in BAL samples fell dramatically within 2 h and remained suppressed for at least 12 h . We conclude that: (1) neutrophils are capable of migration into air spaces after aggregation in pulmonary capillaries in the absence of airway manipulative procedures or instillation of chemoattractants into air spaces and (2) endotoxemia affected surface parameters of PAM in vivo that determine recoverability by lavage. Am J Med Sci, 1984 Jan-Feb, 287(1), 16 - 23 A randomized study of tobramycin plus ticarcillin, tobramycin plus cephalothin and ticarcillin, or tobramycin plus mezlocillin in the treatment of infection in neutropenic patients with malignancies; Lawson RD et al.; Two hundred twenty-five patients with 358 febrile episodes were treated with tobramycin and ticarcillin (TT), tobramycin and mezlocillin (TM), or tobramycin, ticarcillin and cephalothin (TTC) . There were no statistically significant differences in the response rates for patients who were proven to have infection (67% with TT, 69% with TTC and 53% with TM) . Patients were more often cured of their infection if their neutrophil count rose during therapy . In this study, the addition of cephalothin to TT did not increase the frequency of azotemia (10% and 12%, respectively) . Although mezlocillin has a broader spectrum of activity in vitro than ticarcillin, it was not more efficacious when combined with tobramycin than ticarcillin plus tobramycin for the treatment of infections in neutropenic patients. Mol Cell Biol, 1984 Jan, 4(1), 38 - 48 Phenotypic expression in Escherichia coli and nucleotide sequence of two Chinese hamster lung cell cDNAs encoding different dihydrofolate reductases; Melera PW et al.; Nucleotide sequence analysis of two cDNA clones, one shown to direct the synthesis in Escherichia coli of the pI 6.7 form of the 20,000-molecular-weight class of Chinese hamster lung cell dihydrofolate reductase, and the other shown to direct the synthesis of the pI 6.5 form of the 21,000-molecular-weight class of the enzyme, has revealed the following: (i) the differences in physical and enzymatic properties displayed by these two proteins are due to two variations in their respective amino acid sequences with the conversion of Leu to Phe at position 22 probably responsible for the differential sensitivity of these two enzymes to methotrexate and methasquin; (ii) the multiple mRNAs responsible for the synthesis of each of these proteins differ in size due, at least in part, to a length heterogeneity at their 3' ends; (iii) these two proteins are encoded by different genes; and (iv) the sequence AAATATA appears to be a major polyadenylation signal in one Chinese hamster lung cell dihydrofolate reductase gene and a minor signal in another. DNA, 1984, 3(1), 7 - 15 Allele-specific hybridization using oligonucleotide probes of very high specific activity: discrimination of the human beta A- and beta S-globin genes; Studencki AB et al.; The repair activity of Escherichia coli DNA polymerase I (Klenow fragment) was used to prepare nonadecanucleotide hybridization probes which were complementary either to the normal human beta-globin (beta A) or to the sickle cell human beta-globin (beta s) gene . Template-directed polymerization of highly radiolabeled alpha{32P}deoxyribonucleoside triphosphates (dNTPs) onto nonamer and decamer primers produced probes with specific activities ranging from 1.0 X 10(10) to 2.0 X 10(10) dpm/micrograms . The extremely high specific activities of these probes made it possible to detect the beta A and beta S single-copy gene sequences in as little as 1 microgram of total human genomic DNA as well as to discriminate between the homozygous and heterozygous states. Br J Cancer Suppl, 1984, 6, 137 - 43 Implications of repair models for LET effects and other radiobiological phenomena; Alper T; Repair models account for shoulders to survival curves by the postulate of a mode of repair which is depleted ("saturated") as dose increases, and which should therefore be distinguished, conceptually and linguistically, from what is commonly known as "repair of potentially lethal damage" . Acceptance of repair models entails new interpretations of some radiobiological phenomena . "Recovery" of cells between dose fractions would be attributable to reconstitution or resynthesis of the putative agent of repair, so elucidation of the mechanism of such "recovery" requires a different approach from any that have been used in attempts to discover the nature of "sub-lethal lesions" or the mechanism of their repair--attempts that have not been attended by success . Even mammalian cells can yield exponential survival curves; but this fact has been ignored in some proposals for mechanisms of radiation-induced cell killing, and in "theories of RBE" based on multi-sublethal lesion models for shouldered survival curves . According to repair models, however, cells in general are basically single-hit detectors . Comparisons between the responses of repair-proficient cells and their deficient mutants to change in radiation quality support the hypothesis that increases in RBE are attributable to reduced capacity for some mode(s) of repair as LET increases; but there is evidence that some capacity remains, even at very high values of LET. Arch Biochem Biophys, 1984 Jan, 228(1), 133 - 42 Pyruvate formate-lyase (inactive form) and pyruvate formate-lyase activating enzyme of Escherichia coli: isolation and structural properties; Conradt H et al.; The catalytically active form (Ea) of pyruvate formate-lyase in Escherichia coli cells is generated from an inactive form of the enzyme (Ei) through a post-translational process that requires a distinct activating enzyme and is linked to the cleavage of adenosylmethionine to methionine and 5'-deoxyadenosine . Ei and the activating enzyme were purified to homogeneity and structurally characterized . Ei has an alpha 2 oligomeric structure (2 X 85 kDa) and contains no cofactor . The amino acid composition has been determined . Out of a total of six cysteinyl residues per subunit, one shows an unusually fast reaction with iodoacetate (k2 = 7 (M-1 s-1) at pH 6.8, 30 degrees C), which is accompanied by loss of the activatability of the enzyme . The 1500-fold purified activating enzyme is a monomeric protein of 30 kDa . It contains a covalently bound, as yet unidentified chromophoric factor which has an optical absorption peak at 388 nm . Further studies of the in situ state of pyruvate formate-lyase detected a reversible backconversion of the active form Ea into Ei when anaerobic cells become nutrient-depleted. Radiat Res, 1984 Jan, 97(1), 200 - 10 Spectra of base substitution mutations induced in Escherichia coli by tritiated water and the decay of incorporated tritiated thymidine; Ise T et al.; The biological effects of tritiated water and of {6-3H}thymidine or {methyl-3H}thymidine incorporated into DNA were compared with those induced by 60Co gamma rays . The killing efficiencies of tritiated water and the tritium-labeled bases were very similar, between 1.8 and 2.0 in terms of the RBE of 60Co gamma rays when compared with the absorbed dose to the bacterial nucleus . The frequency of His+ revertants induced by the decay of {6-3H}thymidine was 3.5 times higher than that induced by {methyl-3H}thymidine or tritiated water; these revertants were most often the result of A:T leads to G:C transitions . In comparison, the other treatments efficiently induced both transitions and transversions . The mutational spectrum resulting from the decay of tritiated water was also determined in the lacI forward-mutagenesis system of Escherichia coli . Transitions predominated at the low dose (2.5 krad), while both transitions and transversions were recovered after a high dose (18 krad) . These results are very similar to those observed with 60Co gamma rays and are consistent with the hypothesis that mutagenesis resulting from the decay of {6-3H}thymidine is the result of a position effect, while mutagenesis resulting from the decay of {methyl-3H}thymidine and tritiated water is due to beta-particle ionization. Proc Natl Acad Sci U S A, 1984 Jan, 81(2), 424 - 8 Protein HU in the enzymatic replication of the chromosomal origin of Escherichia coli; Dixon NE et al.; A protein that stimulates the enzymatic replication of duplex DNAs of recombinant phages and plasmids bearing the Escherichia coli origin of replication (oriC) has been isolated from an extract of E . coli . The isolated protein and the well-known protein HU, a histone-like DNA-binding protein, have identical polypeptide molecular weights and saturate the oriC replication assay at less than 40 dimers per template DNA circle . This level is one-tenth that needed to coat the template . Protein HU from the blue-green alga Anabaena is similarly active . Antibody specific for protein HU from E . coli inhibits replication promoted both by the reconstituted system and by a crude enzyme extract; in both assays, activity is restored by excess of the isolated protein . Cells lysed in 1 M KCl yield 32,000 dimers of the protein per cell, a number consistent with the reported abundance of HU . These data establish the identity of the isolated factor and protein HU and provide an indication of a function for HU in replication. Proc Natl Acad Sci U S A, 1984 Jan, 81(2), 409 - 13 tRNAMetf2 gene in the leader region of the nusA operon in Escherichia coli; Ishii S et al.; The promoter-proximal portion of the operon containing the Escherichia coli nusA gene has been cloned . Its nucleotide sequence shows that genes for tRNAMetf2 and a 15-kilodalton protein of unknown function precede the nusA protein gene . The sequence suggests that the three genes form a single transcription unit . Consistent with this hypothesis, purified RNA polymerase formed full-length transcripts on the cloned DNA in vitro, although transcription was frequently arrested at the intercistronic site(s) between the gene for tRNAMetf2 and the 15-kilodalton protein. Proc Natl Acad Sci U S A, 1984 Jan, 81(2), 322 - 6 Nuclease protection analysis of ribonucleoprotein complexes: use of the cytotoxic ribonuclease alpha-sarcin to determine the binding sites for Escherichia coli ribosomal proteins L5, L18, and L25 on 5S rRNA; Huber PW et al.; A rapid and convenient method has been devised to determine the binding sites for proteins on RNA . The procedure is an adaptation of one used to map DNA-protein complexes by protection against nuclease digestion . The method uses the cytotoxic ribonuclease alpha-sarcin, which hydrolyzes purines in both single- and double-stranded regions of RNA . It has been authenticated by confirming the binding sites for the Escherichia coli ribosomal proteins L18 and L25 on 5S rRNA and its value has been established by identifying the attachment site for protein L5 . The procedure should be useful for the analysis of other ribonucleoprotein complexes. Proc Natl Acad Sci U S A, 1984 Jan, 81(1), 198 - 201 Evidence for clonal population structure in Escherichia coli; Ochman H et al.; Genotypes of 142 K1 isolates of four O serogroups of Escherichia coli from human hosts in Europe and the United States were characterized by an electrophoretic analysis of allozymic variation in 12 chromosomally encoded enzymes . The genetic structure of natural populations revealed by this analysis is closely similar to that indicated in earlier studies by Achtman and colleagues of the electrophoretic migration pattern for four outer membrane proteins and the chemical structure of the cell-wall lipopolysaccharides . The combined evidence demonstrates that most of the K1 isolates belong to a small number of geographically widespread clones . The distribution of O serogroups among the isolates does not consistently correspond to the clonal structure; O1:K1 isolates represent at least two distantly related, geographically widespread clones, one of which is genetically similar to a clone of the O18:K1 serotype . These findings for K1 isolates add to a growing body of evidence supporting the hypothesis that the genetic structure of natural populations of E . coli is basically clonal, with very limited recombination of chromosomal genes . Clonal structure has important implications for the study of the determinants of pathogenicity and disease specificity in E . coli. Proc Natl Acad Sci U S A, 1984 Jan, 81(1), 185 - 8 RNase III cleavage is obligate for maturation but not for function of Escherichia coli pre-23S rRNA; King TC et al.; RNase III makes the initial cleavages that excise Escherichia coli precursor 16S and 23S rRNA from a single large primary transcript . In mutants deficient in RNase III, no species cleaved by RNase III are detected and the processing of 23S rRNA precursors to form mature 23S rRNA fails entirely . Instead, 50S ribosomes are formed with rRNAs up to several hundred nucleotides longer than mature 23S rRNA . Unexpectedly, these aberrant subunits function well enough to participate in protein synthesis and permit cell growth . Consistent with the inference that RNase III cleavages are absolutely required for 23S rRNA maturation, when 50S ribosomes from a strain deficient in RNase III were incubated with a ribosome-free extract from a RNase III+ strain, rRNA species processed by RNase III and species with normal mature 23S rRNA termini were produced. Proc Natl Acad Sci U S A, 1984 Jan, 81(1), 115 - 9 Location of amino acid alterations in mutants of aspartate transcarbamoylase: Structural aspects of interallelic complementation; Schachman HK et al.; Recent genetic studies of the pyrB locus of Escherichia coli resulted in the characterization of 29 mutant strains harboring defects in the structural gene that encodes the catalytic chains of aspartate transcarbamoylase (carbamoylphosphate: L-aspartate carbamoyltransferase, EC 2.1.3.2) . Three alleles, pyrB554, pyrB730, and pyrB748, have been cloned, and their nucleotide sequences have been determined along with that of the wild-type pyrBI operon in order to locate the sites of the alterations in the catalytic chains . Missense mutation pyrB554 leads to replacement of serine-52 by phenylalanine, and the inactive mutant enzyme has properties similar to those of wild-type aspartate transcarbamoylase . The amber mutation pyrB730 results in unstable truncated polypeptide chains 27 amino acids shorter than wild-type chains . Deletion mutation pyrB748 causes the removal of 181 amino acids . Combining these results with knowledge of the crystallographic structure of the wild-type enzyme provides a basis for tentative structural mechanisms for the observed complementation behavior of the mutant proteins. Mol Gen Genet, 1984, 193(2), 327 - 31 Requirement of protein synthesis for the induction of ribonucleoside diphosphate reductase mRNA in Escherichia coli; Hanke PD et al.; An RNA-DNA hybridization assay was used to quantitate the ribonucleoside diphosphate reductase mRNA synthesis (nrd mRNA) to show that gene expression was dependent on protein synthesis . The increased nrd mRNA synthesis induced by inhibition of DNA synthesis was eliminated by simultaneous inhibition of protein synthesis . It was further found that protein synthesis is required not only initially but continuously during DNA inhibition for increased expression of nrd mRNA synthesis. Mol Gen Genet, 1984, 193(2), 288 - 92 Functional domains of Escherichia coli recA protein deduced from the mutational sites in the gene; Kawashima H et al.; The sites of recA mutations of Escherichia coli, recA441 (tif-1), recA1, recA430 (lexB30) and recA44, were determined by analyses of the nucleotide sequences . All mutations are single point missense mutations within the coding region of the recA gene . In the recA441, recA1, recA430 and recA44 proteins, the 38th, 160th, 204th, and 246th amino acids, respectively, from the amino terminal ends are altered . Based on the properties of these mutant proteins and modified forms of recA protein, the locations of various regions of the recA protein that are involved in binding with ATP, binding with single-stranded DNA, hydrolysis of ATP, interaction between the recA protein molecules and interaction with the lambda cI or lexA repressors are mapped on the primary structure of the protein. Mol Gen Genet, 1984, 193(2), 210 - 3 Autoregulation of the rho gene of Escherichia coli K-12; Kung H et al.; It has previously been proposed, based on indirect evidence, that the Rho protein may control the expression of the rho gene . Using an in vitro system for the transcription and translation of the rho gene cloned into plasmid pBR322, we tested this hypothesis directly by monitoring the effect in vitro of excess or limiting Rho protein . The addition of purified Rho protein suppresses Rho synthesis in vitro . The addition of antibody to Rho specifically stimulates Rho synthesis in vitro . The stimulation of Rho factor synthesis by antibody to Rho is reversed by Rho protein . Rho factor purified from a strain with a mutationally altered rho gene (rho-115) does not suppress Rho synthesis in vitro . These results provide convincing evidence that the rho gene is subject to autoregulation. Ann Surg, 1984 Jan, 199(1), 37 - 43 The adjuvant effect of peritoneal fluid in experimental peritonitis . Mechanism and clinical implications; Dunn DL et al.; At laparotomy, many surgeons routinely instill crystalloid solutions into the peritoneal cavity, presumably to dilute out necrotic debris, bacteria, and adjuvant substances which foster bacterial growth . We examined the effect on mortality, bacterial growth, clearance, and phagocytosis of various volumes of saline instilled into the peritoneal cavity of rats during Escherichia coli peritonitis . Minimal intraperitoneal bacterial growth was seen after the introduction of a nonlethal inoculum of viable E . coli in 1 ml of saline, while administration of an identical inoculum in 30 ml of saline intraperitoneally (i.p.) led to increased 48-hour mortality (p less than 0.01), and associated rapid bacterial proliferation (p less than 0.01) . Clearance of nonviable radiolabelled E . coli from the peritoneal cavity was delayed, bacterial association with host peritoneal leukocytes was decreased, and blood uptake of radiolabelled bacteria was diminished in animals receiving 30 ml of saline i.p., compared to controls which received the identical inoculum in 1 ml of saline i.p . The clinical relevance of these studies is manifold: (1) they provide a possible explanation why patients with ascites due to cirrhosis or the nephrotic syndrome, or those patients undergoing peritoneal dialysis are more susceptible to primary and secondary bacterial peritonitis, possibly on the basis of impaired peritoneal clearance or diminished phagocytosis and, (2) although irrigation of the peritoneal cavity with crystalloid solution would seem prudent during laparotomy, these solutions must be removed prior to closure to prevent interference with normal peritoneal host defense mechanisms. Urology, 1984 Jan, 23(1), 58 - 61 Xanthogranulomatous pyelonephritis in renal transplant recipient; Carson CC et al.; Xanthogranulomatous pyelonephritis is reported in the native kidney of a renal allograft recipient . Immunoglobulin deposition in the transplant kidney in the absence of cell-mediated rejection, accompanied by selective cultures showing Escherichia coli from the native kidney, led to the diagnosis . Native nephrectomy resulted in resolution of the patient's chronic bacteriuria and creatinine elevation. Mol Gen Genet, 1984, 193(1), 8 - 16 Promoter selectivity of Escherichia coli RNA polymerase . II: Altered promoter selection by mutant holoenzymes; Nomura T et al.; Using the in vitro mixed transcription system (Kajitani and Ishihama (1983a, 1983b), we examined selective transcription of truncated DNA templates carrying lac(UV5), rrnE or rpsA promoters by RNA polymerase holoenzymes from pairs of wild-type parents and mutants with a mutation in one or more RNA polymerase subunit genes . The promoter selectivity of RNA polymerases from two sigma-subunit mutants carrying either rpoD2 or rpoD285 differed markedly from that of the respective wild-type enzymes . Both the parental RNA polymerases, however, exhibited abnormal promoter selectivity compared with holoenzymes from various wild-type E . coli strains . On the other hand, all the RNA polymerases from rpoB and/or rpoC mutants and the respective wild-type parents were similar, if not identical, in promoter selection at low temperature . At high temperature, however, RNA polymerases from mutants carrying rpoB2B7 and rpoC4, affecting the beta and beta' subunits, respectively, showed decreased transcription from the high-affinity slow-transcribable promoter rrnEp2 whereas the rpoC92 and rpoB906 X rpoC907 mutant enzymes both lost transcription activity from the strong promoter lacP(UV5) . Taking all these observations together we conclude that not only the sigma subunit but also the beta and beta' subunits are involved in the recognition of promoters. Mol Gen Genet, 1984, 193(1), 76 - 81 Missense and nonsense suppressors derived from a glycine tRNA by nucleotide insertion and deletion in vivo; Murgola EJ et al.; Beginning with a missense suppressor tRNA and a nonsense suppressor tRNA, both in Escherichia coli and each containing an extra nucleotide in the anticodon loop, we generated new suppressors in vivo by spontaneous deletion of specific nucleotides from the anticodon loop . In one experiment, the new suppressor was generated by a double mutational event, base substitution and nucleotide deletion . A novel ochre suppressor is also described . It is very efficient in nonsense suppression but has no ms2i6 modification of the A residue on the 3' side of the anticodon . The results have important implications for tRNA structure-function relationships, tRNA recognition by tRNA-modifying enzymes, mechanisms of deletion mutation, and tRNA evolution. J Virol, 1984 Jan, 49(1), 273 - 5 Requirement for a fluid host cell membrane in injection of coliphage T5 DNA; Labedan B; Injection of T5 first-step-transfer DNA was prevented at 29 degrees C, after adsorption to an unsaturated fatty acid mutant grown on elaidate (phase transition at 35 degrees C) . Local anesthetics, which increase membrane fluidity, did not inhibit injection above transition temperature and could even reverse the inhibition observed at 29 degrees C on elaidate cells. J Virol, 1984 Jan, 49(1), 132 - 41 Analysis of adenovirus transforming proteins from early regions 1A and 1B with antisera to inducible fusion antigens produced in Escherichia coli; Spindler KR et al.; Plasmid vectors were constructed which expressed three adenovirus tumor antigens fused to a portion of the trpE protein of Escherichia coli . Insertion of adenovirus type 2 DNA from early region 1A (E1A) into such a plasmid led to a fusion protein which contained the C-terminal 266 amino acids of the 289-amino acid protein encoded by the viral 13S mRNA . Similarly, insertion of adenovirus type 5 DNA corresponding to the E1B 55- and 21-kilodalton proteins led to production of fusion proteins containing amino acid sequences from these proteins . After induction with indoleacrylic acid, fusion proteins accumulated stably in the E . coli cells . By using a simple extraction of insoluble protein, 1 to 10 mg of fusion protein per liter of culture was obtained . The fusion proteins were purified on preparative polyacrylamide gels and used to immunize rabbits . Specific antisera for the E1A 289- and closely related 243-amino acid proteins and the E1B 55- and 21-kilodalton proteins were obtained . These sera were used to immunoprecipitate the tumor antigens in cells infected with wild-type and various mutants of adenovirus or to analyze them by an immunoblotting procedure . Mutant E1A proteins in which the C-terminal 70 amino acids are deleted were phosphorylated to much lower extents than the wild-type E1A proteins . This indicates that the deleted region is important for the process of phosphorylation . The E1A proteins were extracted, sedimented in glycerol gradients, analyzed by immunoprecipitation, and found to sediment primarily as monomers. J Bacteriol, 1984 Jan, 157(1), 35 - 8 Induction by UV light of the SOS function sfiA in Escherichia coli strains deficient or proficient in excision repair; Quillardet P et al.; The influence of the nucleotide excision repair system on the induction by UV irradiation of the SOS function sfiA has been investigated . The level of sfiA expression was monitored by means of a sfiA::lacZ operon fusion in both the wild-type strain and a uvrA mutant . We found that the initial steady rate of sfiA expression was proportional to the UV dose and was identical in uvr+ and uvrA backgrounds . This suggests that the initial steady rate of sfiA expression is determined by the initial number of lesions and before any effect of excision repair . We confirmed that after 2 h of expression the net synthesis of sfiA product is, for the same UV dose, about five times lower in uvr+ than in uvrA strains . We show that this is due to earlier repression of the SOS system in uvr+ than in uvrA strains and not to different initial rates. J Bacteriol, 1984 Jan, 157(1), 324 - 6 Cloning of a Thiobacillus ferrooxidans plasmid in Escherichia coli; Holmes DS et al.; Three separate plasmids of 6, 7, 16, and greater than 23 kilobases were purified from a single clone of Thiobacillus ferrooxidans ATCC 33020 grown in the presence of uranium . The 6.7-kilobase plasmid (pTf1) was cloned separately into the HindIII or BamHI site of Escherichia coli plasmid pBR322 . Restriction maps of the recombinant plasmids, termed pTf100 and pTf110, respectively, were constructed, creating potential cloning vehicles for exchanging genetic information between E . coli and T . ferrooxidans . Evidence from restriction enzyme analysis and Southern blot DNA-DNA hybridization indicates that the three native plasmids share little sequence homology. J Bacteriol, 1984 Jan, 157(1), 247 - 52 Gene regulation in plasmid RK2: positive control by korA in the expression of korC; Young C et al.; The broad-host-range plasmid RK2 encodes three host-lethal kil genes whose actions are controlled by specific kor genes . We have shown previously that the 0' to 5.5' region of RK2 encodes both kilA and korC . Because of the lethal effect of kilA, plasmids with this region cannot be maintained in Escherichia coli unless the RK2 korA gene is also present . To investigate korC in the absence of kilA and therefore of korA, we first mapped kilA and korC to specific segments of the cloned 0' to 5.5' region . This allowed us to construct a korC+ plasmid missing the kilA region and thereby removed the need to have korA in the cell . We found that this korC-encoding plasmid alone is insufficient to control kilC . The korA function is required, and it can be supplied in trans . We also constructed a kilA+ korC- plasmid and found that korA is sufficient to control kilA . Thus, in addition to acting negatively to control kilA, korA acts positively to allow korC control of kilC . This korA dependence of korC is bypassed in a rho-115 mutant of E . coli . We consider the possibility that korA product acts as an antiterminator of transcription in korC expression. J Bacteriol, 1984 Jan, 157(1), 233 - 9 Effects of ethanol on the Escherichia coli plasma membrane; Dombek KM et al.; The effects of ethanol on the fluidity of Escherichia coli plasma membranes were examined by using a variety of fluorescent probes: 1,6-diphenyl-1,3,5-hexatriene, perylene, and a set of n-(9-anthroyloxy) fatty acids . The anthroyloxy fatty acid probes were used to examine the fluidity gradient across the width of the plasma membrane and artificial membranes prepared from lipid extracts of plasma membranes . Ethanol caused a small decrease in the polarization of probes primarily located near the membrane surface . In comparison, hexanol decreased the polarization of probes located more deeply in the membrane . Temperature had a large effect on probes located at all depths . The effects of ethanol on E . coli membranes from cells grown with or without ethanol were also examined . Plasma membranes isolated from cells grown in the presence of ethanol were more rigid than those from control cells . In contrast to plasma membranes, artificial membranes prepared from lipid extracts of ethanol-grown cells were more fluid than those from control cells . These differences are explained by analyses of membrane composition . Membranes from cells grown in the presence of ethanol are more rigid than those from control cells due to a decrease in the lipid-to-protein ratio . This change more than compensates for the fluidizing effect of ethanol and the ethanol-induced increase in membrane C18:1 fatty acid which occurs during growth . Our results suggest that the regulation of the lipid-to-protein ratio of the plasma membrane may be an important adaptive response of E . coli to growth in the presence of ethanol. J Bacteriol, 1984 Jan, 157(1), 184 - 9 Purification and subunit composition of acetohydroxyacid synthase I from Escherichia coli K-12; Eoyang L et al.; Acetohydroxyacid synthase I from Escherichia coli K-12 has been purified to near homogeneity . Analysis of the purified enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of two polypeptides, one with a molecular weight of 60,000 and one with a molecular weight of 9,500 . These two polypeptides were present in constant proportion to each other and to enzyme activity . The molar ratio of the two polypeptides (Mr 9,500:60,000), estimated from stained polyacrylamide gels, was 1 . Antisera raised against the 60,000 Mr polypeptide precipitated both the 60,000 and the 9,500 Mr polypeptides from extracts of cells labeled with {35S}methionine . The addition of sodium dodecyl sulfate before immunoprecipitation eliminated the smaller polypeptide, and only the larger one was recovered . The hydrodynamic properties of the native enzyme confirmed a previous report that the largest enzymatically active species has a molecular weight of about 200,000; this species contains both the 60,000- and 9,500-molecular-weight polypeptides. Infect Immun, 1984 Jan, 43(1), 257 - 62 Inhibition of blood clearance and hepatic tissue binding of Escherichia coli by liver lectin-specific sugars and glycoproteins; Perry A et al.; The effects of sugars and glycoproteins that are known to bind to lectins of liver tissue on the clearance of cells of Escherichia coli from mouse blood was investigated . The administration of 100 mg per mouse of methyl-alpha-D-mannoside, methyl-alpha-D-glucoside, or methyl-alpha-D-fucoside, but not of methyl-alpha-D-galactoside or L-rhamnose, markedly inhibited the blood clearance of cells of E . coli 346 . Clearance was similarly inhibited by 0.1 and 1.0 mg per mouse of asialofetuin or ovalbumin, respectively, whereas fetuin had no effect . The inhibitory effects of the sugars on blood clearance was abolished by pretreating the E . coli cells with antibodies against whole organisms . All of these effects were equal for fimbriated and nonfimbriated phenotypes of E . coli 346 . Homogenates of mouse liver tissue coaggregated with nonfimbriated cells of E . coli . The aggregation was blocked by 100 mM solutions of methyl-alpha-D-mannoside, or methyl-alpha-D-glucoside, 1 mg of bacterial lipopolysaccharide per ml, or 10 mM EDTA but not by L-rhamnose . These results suggest that the mannose-N-acetylglucosamine hepatic lectin recognizes specific sugars on the surface of E . coli and may be centrally involved in the nonimmune clearance of nonfimbriated E . coli from the blood of the infected host. Arch Surg, 1984 Jan, 119(1), 71 - 4 Effect of abdominal operations on survival after septic challenge in normal rats; Selivanov V et al.; We evaluated the effect of 60% small-bowel enterectomy that removed 75% of Peyer's patches, and the effects of lesser abdominal operations, on the survival of rats following inducement of peritonitis . We divided 286 female Fischer rats weighing 140 to 200 g into four experimental and two control groups . Following enterectomy, rats recovered for ten to 14 days before peritonitis was induced by intraperitoneal injection of 0.7 mL/100 g of body weight of a solution of 4% hemoglobin and Escherichia coli (10(9)/mL) . At 48 hours after enterectomy, the survival rate was better than that in rats not operated on or anesthetized . Survival rates for rats having lesser intra-abdominal operations were significantly greater after peritonitis than the rates for controls, but were similar to rates for rats having enterectomies . Our results suggest that intra-abdominal operation of minimal or large magnitude is associated with improved survival from hemoglobin-E coli adjuvant peritonitis. Schweiz Med Wochenschr Suppl, 1984, 17, 7 - 11 {Epidemiology of travel diarrhea}; Steffen R; Travelers' diarrhea is the most frequent health problem during a stay in developing countries . A recent study basing on interviews with 16,568 charter flight passengers returning to Europe from 13 destinations in various climatic regions provides epidemiological data on a worldwide scale . Significant differences in diarrheal incidence varied not only between individual destinations, but also between hotels in the same area . The highest incidence for a two weeks' stay exceeded 50% in some regions of North and West Africa . Persons under 30 were more often affected than older travelers . Within international groups meeting in developing countries, the risk varied according to the patient's country of origin, with the residents of industrialized nations being most often affected . Even in the tropics, diarrhea usually takes a short and mild course . The various regions show unessential differences in chronology and symptomatology . E . coli is the most frequent causative agent of this ailment. Arch Virol, 1984, 81(1-2), 67 - 78 Molecular cloning of the genomes of poliovirus type 3 strains by the cDNA: RNA hybrid method; Stanway G et al.; The genomes of two neurovirulent strains of poliovirus type 3, wild type P3/Leon/37 and a vaccine revertant P3/119/70, have been cloned in E . coli . The cDNA: RNA hybrid method used was efficient and may have wide applicability for cDNA cloning . Overlapping clones spanning the entire genome were obtained for each strain . These have been used to produce full-length DNA copies of the two genomes each within a single plasmid. Mol Gen Genet, 1984, 194(3), 457 - 65 Molecular cloning of the yeast fatty acid synthetase genes, FAS1 and FAS2: illustrating the structure of the FAS1 cluster gene by transcript mapping and transformation studies; Schweizer M et al.; From a Saccharomyces cerevisiae gene bank contained in the novel yeast cosmid shuttle vector pMS201 the fatty acid synthetase (FAS) genes FAS1 and FAS2 were isolated . FAS clones were identified by in situ colony hybridization using two yeast DNA probes apparently capable of producing avian FAS cross-reacting material (J . Carbon, personal communication) . Classification as FAS1 or FAS2 clones was achieved by their specific transformation of fas1 and fas2 yeast mutants . By transcription mapping FAS1 was assigned to about 5.3 kb within 14.8 kb of chromosomal DNA covered by two genomically adjacent BamHI fragments . The FAS2 gene was localized on a single BamHI fragment of 25 kb . One of the FAS clones ( FAS2 ) produces immunologically cross-reacting material in Escherichia coli . High frequency transformation of fas1 mutants was only observed with one subclone, pMS3021 , containing the intact FAS1 locus . Other DNA segments cloned in the same self-replicating vector but representing only part of FAS1 exhibited drastically lower transformation rates . As evident from this and from FAS1 /TRP1-cotransformation rates only the intact FAS1 gene in pMS3021 is capable of fas1 -mutant complementation . With partial FAS1 genes, even when coding for an intact equivalent of the mutated domain, their chromosomal integration is necessary for the expression of FAS . In integrative transformants the coexistence of integrated and autonomously replicating plasmid DNA was demonstrated . Both, the extrachromosomal and chromosomally integrated FAS DNA was mitotically unstable . Transformation studies using subcloned FAS1 DNA segments revealed the relative locations of the enoyl reductase and dehydratase domains within this pentafunctional cluster gene. Mol Gen Genet, 1984, 194(3), 444 - 50 Does Tn10 transpose via the cointegrate molecule? Harayama S, Oguchi T, Iino T. It has been well established that Tn3 and its relatives transpose from one replicon to another by two successive reactions: formation of the cointegrate molecule and resolution from it . Whether or not the 9300 base pair tetracycline resistance transposon Tn10 transposes in the same manner as Tn3 was investigated by two methods . In the first method, lambda 55, a lambda phage carrying Tn10 was lysogenized in an Escherichia coli strain carrying a Tn10 insertion; the phage has a deletion in attP, hence it was lysogenized in a Tn10 sequence in the E . coli chromosome by reciprocal recombination . The chromosomal structure in these lysogens is equivalent to the Tn10-mediated cointegrate molecule of lambda and the E . coli chromosomal DNA . The stability of the cointegrate molecule was examined by measuring the rate of excision of lambda from the host chromosome, and was found to be stable, especially in a Rec- strain . Because of this stability, the cointegrate molecule should be accumulated if Tn10 transposes via the cointegrate molecule . Then, we examined the configuration of products made by transposition of Tn10 from lambda 55 to the E . coli chromosome . The cointegrate molecule was found in products of Tn10 transposition in a Rec+ strain at a frequency of 5% per Tn10 transposition, but this molecule could not be found in a Rec- strain . Since transposition of Tn10 was recA-independent, absence of the cointegrate molecule formed in a RecA- strain strongly suggested that the cointegrate molecule is not an obligatory intermediate of transposition of Tn10.(ABSTRACT TRUNCATED AT 250 WORDS) Mol Gen Genet, 1984, 194(3), 410 - 5 Cloning and characterization of the xyl genes from Escherichia coli; Rosenfeld SA et al.; Specific xylose utilization mutants of Escherichia coli were isolated that had altered xylose isomerase ( xylA ), xylulokinase ( xylB ), and regulatory ( xylR ) or transport ( xylT ) activities . We screened the Clarke and Carbon E . coli gene bank and one clone, pLC10 -15, was found to complement the xyl mutants we had characterized . Subcloning and DNA restriction mapping allowed us to locate the xylA and xylB genes on a 1.6 kbp Bg/II fragment and a 2.6 kbp HindIII-Sa/I fragment, respectively . The identification and mapping of xyl gene promoters suggest that the xylA and xylB genes are organized as an operon having a single xylose inducible promoter preceding the xylA gene. Mol Gen Genet, 1984, 194(3), 388 - 94 lacZ fusions to genes that specify exported proteins: a general technique; Palva ET et al.; We have devised a general, one-step technique for isolation of strains in which the gene coding for an exported protein is fused to the gene for beta-galactosidase (lacZ) . These fusions specify a hybrid protein comprised of an NH2-terminal portion of the exported protein and a large functional COOH-terminal portion of beta-galactosidase . The fusions are constructed with a derivative of the MudII (lac, Ap) phage . To overcome the lethality that is often associated with the expression of such a hybrid gene, we have recombined an early lacZ nonsense mutation onto this phage . With the use of strains that carry a temperature-sensitive nonsense suppressor, expression of the full-length hybrid protein can be controlled by varying the growth temperature . We demonstrated the utility of this technique by isolating a series of fusions to a gene, ompA, coding for a major outer membrane protein . As expected, strains containing these fusions are not viable under conditions that permit synthesis of a functional nonsense suppressor . Accordingly, this method should also be useful for direct selection of export-defective mutants. Adv Exp Med Biol, 1984, 172, 295 - 318 Cloning of the rat endogenous helper leukemia virus DNA sequence and expression of the helper activity encoded by the cloned DNA sequence in normal rat kidney cells by microinjection; Yang SS et al.; By the use of recombinant DNA technology and microinjection in cultured cells, the molecular genetic elements involved in the evolution of a retrovirus with the multipotential to infect, transform and replicate in host cell, have been critically examined in this investigation . Recently we have identified and purified the integrated and proviral DNA sequences specific for two rat endogenous helper leukemia viruses, WR- RaLV , originated from a chemically induced wild rat fibrosarcoma, and RHHV , isolated from a chemically induced rat hepatoma, HTC-H1 (1) . By using a multidisciplinary approach combining restriction endonuclease analysis, reverse phase V-column chromatography, agarose gel electrophoresis, Southern blot transfer and filter nucleic acid hybridization, we were able to demonstrate that the rat helper leukemia viral DNA sequence was approximately 8.4-8.8 kb . The 8.8 kb RHHV DNA was molecularly cloned via the EK-1 certified vector pBR 322 plasmid into E . coli RRI cells . A successful recombinant clone, 8/32, that carried one entire RHHV 8.8 kb DNA sequence was mapped by restriction endonuclease analyses . Restricted DNA fragments of various sizes throughout the complete RHHV genome were isolated and purified for intranuclear microinjection into normal rat kidney cells . Release of type C infectious helper virus in these microinjected cells was investigated by superinfection on K-NRK, Kirsten sarcoma transformed non-producer cells . Recombination of the helper viral DNA sequence, en toto or of subgenomic sizes, carried in microinjected cells, with the sarcomagenic DNA sequence, carried in K-NRK cells, was also studied by genome-rescue and cell-transformation experiments . Our observations led to the conclusion that all critical genetic elements including the 5' LTR helper DNA sequence, gag, pol, and env genes, encoded for the biological activity of the type C helper virus resided within the 6.0 kb proximal to the 5' terminus of the endogenous rat type C helper virus DNA . They proved vitally essential for the recombination with the Src sequence during the evolution of an infectious, transforming and replication-competent retrovirus. Mol Gen Genet, 1984, 194(1-2), 60 - 4 Mu DNA replication in vitro: criteria for initiation; Higgins NP et al.; An in vitro system for investigating Mu replication nd transposition using film lysates has recently been described (Higgins et al . 1983) . Under most conditions examined, little or no replication initiation takes place in vitro . The data are consistent with Mu specific replication forks being initiated in vivo, and completing but not reinitiating a round of replication in vitro . Since Mu DNA replication is from left to right, an excess of right end sequences compared to left end sequences are replicated on the film lysates . Two conditions reported to specifically decrease Mu DNA replication in vivo ( Pato and Reich 1982) were assessed for their effects on in vitro replication . Protein synthesis inhibition in vivo drastically decreased Mu specific DNA synthesis both in vivo and in the film lysates . However, temperature-sensitive (ts) A cells (A ts) incubated at the non-permissive temperature gave increased Mu synthesis at the permissive temperature in vitro . These conditions result in preferential mobilization of Mu specific forks, equal replication of the left and right end sequences of Mu, and meet minimal criteria for Mu replication initiation in the Ats lysates . The results are consistent with the Mu A protein limiting the initiation of Mu replication in vitro. Mol Gen Genet, 1984, 194(1-2), 322 - 9 Genetic analysis and molecular cloning of the Escherichia coli ruv gene; Shurvinton CE et al.; The genetic organisation of the ruv gene, a component of the SOS system for DNA repair and recombination in Escherichia coli, was investigated . New point mutations as well as insertions and deletions were generated using transposon Tn10 inserted in eda as a linked marker for site specific mutagenesis, or directly as a mutagen . The mutations were ordered with respect to one another and previously isolated ruv alleles by means of transductional crosses . The direction of chromosome mobilization from ruv ::Mud( ApRlac ) strains carrying F42lac + established that ruv is transcribed in a counterclockwise direction . Recombinant lambda phages able to restore UV resistance to ruv mutants were identified,and the ruv + region was subcloned into a low copy number plasmid . The ruv + plasmid was able to correct the extreme radiation sensitivity and recombination deficiency of ruv recBC sbcB strains. Mol Gen Genet, 1984, 194(1-2), 241 - 7 Identification of the dye gene product, mutational loss of which alters envelope protein composition and also affects sex factor F expression in Escherichia coli K-12; Buxton RS et al.; The product of the dye gene of Escherichia coli, mapping at 99-100 min, is required for expression of the sex factor F, and also appears to be involved in the regulation of envelope proteins . Mutation of dye thus results in loss of expression of the F-factor ( Fex -), i.e . male sterility, and dye sensitivity (Dyes) . We have isolated a plasmid, pRB38 , in which a 6 kb SalI fragment carrying the dye+ gene was cloned into the plasmid pACYC184 . This 6 kb SalI fragment also carries two nearby markers, chlG , involved in the synthesis of the molybdenum cofactor, and phoM, required for constitutive expression of alkaline phosphatase . Some of the polypeptides synthesised by pRB38 were identified using the maxi-cell procedure . The product of the dye gene was found to be a polypeptide of Mr = 29,000 . Thus derivatives of pRB38 in which the transposon gamma delta was inserted into dye, resulting in a Dyes Fex - phenotype when these plasmids were in a delta dye strain, failed to a produce this polypeptide and in some cases produced a truncated product . Such insertions also resulted in a Chlr and Pho- phenotype when the plasmid was in a delta (dye- chlG -phoM) phoR strain, although complementation tests suggested that the phoM+ and chlG + genes were still intact . Insertions of gamma delta into the promoter distal end of dye did not result in a Dyes Fex - phenotype, although a truncated Dye protein was synthesised, and a Chlr Pho- phenotype was produced . It has been suggested ( Gaffney et al . 1983) that the dye (= sfrA ) gene product is necessary for F-factor expression because it is required for translocation of the F-factor TraJ protein to the outer membrane.(ABSTRACT TRUNCATED AT 250 WORDS) Mol Gen Genet, 1984, 194(1-2), 206 - 10 IS8- and Tn2353-mediated cointegration of the plasmids R15 and RP4::Tn1; Dobritsa AP; The plasmids R15 and RP4::Tn1 form fused structures (85 Md and 92 Md cointegrates) . The cointegrates do not resolve practically in recA- Escherichia coli cells and have a mean life-time of more than 50 generations in a recA+ background . The 85 Md cointegrates were generated at a frequency of 4 x 10(-4) per R15 transconjugant during a mating between E . coli {R15; RP4::Tn1} and E . coli {F' ColVBtrp :: Tn1755 } . These plasmids carry two directly repeated copies of the mobile element IS8 at the junctions between R15 and RP4::Tn1 . The transposition of IS8 from RP4::Tn1 to the R15 plasmid and the formation of hybrid molecules promoted by this process appear to be induced by the IS8 element of the Tn1755 structure during or after conjugal transfer of F' ColVBtrp :: Tn1755 into E . coli {R15; RP4::Tn1} cells . The formation of the 92 Md cointegrates occurs at a frequency of 2 x 10(-5) . The fused molecules of R15 and RP4::Tn1 carry two direct copies of an 8.65 Md R15 fragment at the junctions between these replicons . The fragment has specific features of a new transposon . This element designated Tn2353 determines resistance to Hg, Sm and Su and contains two sites for each BamHI, Bg/II and Sa/I and three sites for both EcoRI and PstI . The physical map and some other characteristics of Tn2353 are presented. Mol Gen Genet, 1984, 194(1-2), 179 - 87 Insertions of transposable elements in the promoter proximal region of the gene cluster for Escherichia coli H+-ATPase: 8 base pair repeat generated by insertion of IS1; Kanazawa H et al.; A plasmid pKY159 (Yamaguchi and Yamaguchi 1983) carrying a promoter proximal portion of the gene cluster of the proton-translocating ATPase (H+-ATPase) of Escherichia coli causes growth inhibition of wild-type cells . Insertion of a transposable element in this plasmid released this inhibitory effect . In analyzing this inhibitory effect, we determined the insertion points at the nucleotide-sequence level of transposable elements on 30 independent derivatives of pKY159 . Insertions of IS1, IS5, and gamma delta were found between the promoter and the gene for a possible component of 14,000 daltons of the H+-ATPase . Of 31 insertions, 26 were of IS1 and were located at the same site, indicating that this site is a hotspot for IS1 insertion and that IS1 insertion is much more frequent than that of IS5 or gamma delta in this region . Four different sites for IS1 insertion were found; in two of these an 8 base pair (bp) duplicate of the target sequence ( AAAAACGT and AAACGTTG ) was generated, while in the other two a 9 bp duplicate was found . In all cases in this study the nucleotide sequence of IS1 was the same as that of IS1-K . In the two cases with an 8 bp duplicate in different sites, a common 6 bp sequence ( AAACGT ) was found . These results suggested that generation of the 8 bp duplicate is related to the common sequence rather than a mutation in IS1 suggested by Iida et al . (1981) and also suggested that the essential length of the duplicate is 8 bp or less than 8 bp . A 6 bp sequence ( GTGATG ) homologous to the end portion of IS1 was found at the hotspot , but not at other sites, suggesting that this homology contributed to the high frequency of IS1 insertion.(ABSTRACT TRUNCATED AT 250 WORDS) Folia Biol (Praha), 1984, 30 Spec No, 93 - 104 Local stability involved in characterizing and controlling promoters in eukaryotes; Gabarro-Arpa J et al.; Eukaryotic promoters with known in vivo activities have been analysed for characteristic stability patterns . Correlation of transcription yield in promoter mutants with size and stability of individual domains of the promoter stability profile supports the conclusion that eukaryotic promoters are built up by at least three elements: a region enabling the transcription ("enhancer"), with a characteristic stability pattern; an activator domain, with high GC content, whose activator potential is controlled by the domain stability and length; a trap domain, with high AT content, setting the cap site . The activated enzyme undergoes a steady deactivation process, losing half of its activity upon moving 55 bases between the activator and the trap site. Folia Biol (Praha), 1984, 30 Spec No, 7 - 17 The structure and expression of the mercury-resistance transposon Tn501; Brown NL; The nucleotide sequence of the mercury-resistance transposon Tn501 has been determined and the functional regions have been identified by a variety of techniques . The transposition gene products are structurally and functionally homologous to those of other transposons in the Tn3 family and of other recombination systems . The expression of the transposition genes may be regulated coordinately with that of the mercury-resistance genes . Comparison of related transposons suggests mechanisms for their evolution. Avian Dis, 1984 Jan-Mar, 28(1), 147 - 53 Pathogenicity of various adenovirus serotypes in the presence of Escherichia coli in chickens; Dhillon AS et al.; Ten strains of adenovirus representing 10 serotypes were administered intratracheally to 3-week-old specific-pathogen-free chickens, which also received 2.9 X 10(5) colony-forming units of a pathogenic Escherichia coli intranasally . One group was given only E . coli, and one was retained as an uninoculated control . Gross pathologic alterations post-mortem were minimal and limited to multiple scattered, pale areas in the lungs of an occasional chicken in various groups . Histopathologic changes in the lungs were those of multifocal, interstitial, and occasionally diffuse pneumonia . Moderate to marked interstitial pneumonia was incited by adenovirus strains 75-1A, B-3 A-2, C-2B, and X-11; Ind-C, Stein, Tipton, J-2, and T-8 caused similar but milder lesions . Strains 75-1A, A-2, C-2B, T-8, and X-11 incited moderate to marked multifocal pneumonia; Ind-C, Stein, Tipton, J-2, and B-3 caused mild multifocal pneumonia . In all groups, the pneumonic lesions were more severe 5 days postinoculation than 12 days postinoculation . Bronchiolitis and tracheitis lesions also varied in severity with serotype . A mild hepatitis was seen with serotypes T-8 and 75-1A . Neither the uninoculated control group nor the group inoculated with only E . coli exhibited gross or histopathologic alterations. Gene, 1984 Jan, 27(1), 87 - 99 Cloning of eukaryotic genes in single-strand phage vectors: the human interferon genes; Bowden DW et al.; Using oligonucleotide probes with defined sequences, we have selected clones from a human lymphocyte cDNA library which represent human leukocyte (HuIFN-alpha) and fibroblast (HuIFN-beta) interferon gene sequences . Double-stranded f1 phage DNA was used as the vector for initial cloning of cDNA . Clones carrying interferon gene sequences were identified by hybridization with the oligonucleotide probes . The same oligonucleotide probes were used as primers for dideoxy chain termination sequencing of the clones . One HuIFN-alpha clone, 201, has a nucleotide sequence different from published HuIFN-alpha sequences . Under control of the lacUV5 promoter, the 201 gene has been used to express biologically active HuIFN-alpha in Escherichia coli. Circ Shock, 1984, 12(3), 177 - 89 Role of angiotensin I and glucagon in canine endotoxin shock: effect of converting enzyme inhibitor and prior immunization; Manson NH et al.; he intermediate and latter stages of canine endotoxin shock are characterized by a progressive decrease in cardiac output, increase in total peripheral resistance, and hypoglycemia . We have hypothesized that the renin-angiotensin system and glucagon may mediate the loss of cardiovascular and glucose homeostasis . E . Coli endotoxin shock (1 mg/kg; 055:B5) was induced in three groups of dogs and systemic hemodynamics, angiotensin I activity, and glucagon were monitored for 5 hr; endotoxin shock (n = 13); endotoxin shock + prior immunization with J5 mutant of E coli 0111 (n = 5); Endotoxin + captopril (20 micrograms/kg/hr; n = 9); and sham-operated time-matched controls (n = 8) . Thirty minutes postshock, angiotensin I and glucagon began to increase . Angiotensin I activity reached a peak at 60 min postendotoxin (90 +/- 25 vs 5 +/- ng/ml/hr; p less than 0.001) and plateaued . Increased glucagon levels plateaued at 3.5 hr postshock (1500 +/- 200 vs 155 +/- 77 pg/ml; p less than 0.001) . Cardiac output began to progressively decrease, total peripheral resistance began to increase, and persistent hypoglycemia developed at 3 hr postshock . Captopril inhibited the increase in total peripheral resistance and had no effect on the decrease in cardiac output or the hypoglycemia . The initial glucagon response was attenuated but there was no difference at 5 hr (950 +/- 150 vs 1200 +/- 200 pg/ml) . Prior immunization significantly preserved cardiac output, total peripheral resistance, plasma glucose levels, glucagon levels, and angiotensin I activity . It is concluded that 1) the renin-angiotensin system is a physiologic and not a pathophysiologic compensatory mechanism during the course of endotoxin shock and that inhibition of this system is deleterious; 2) glucagon may serve as an important mediator of both the myocardial dysfunction and glucose dyshomeostasis of endotoxin shock; and 3) immunological inhibition of the initial phase of endotoxin shock significantly preserves cardiovascular and glucose homeostasis and adds support to the concept that the initial vascular phase of endotoxin shock plays a primary role in determining the severity of the endotoxin/septic shock syndrome. Plasmid, 1984 Jan, 11(1), 96 - 8 Physical and functional map of the hemolytic plasmid pSU316; Andres I et al.; A restriction endonuclease analysis of the hemolytic plasmid pSU316 has allowed location of the cleavage sites for the endonucleases BamHI, XbaI, KpnI, Bg/II, Sa/GI, EcoRI, and HindIII . Hybridization experiments between pSU316 and pED100 have shown that the tra region of pSU316 lies in a segment comprising part of Sa/GI fragments S-1 and S-3 and the entire fragment S-4 . The positions of other plasmids coded functions, namely the replication functions and alpha-hemolysin production, have been determined in the physical map. Plasmid, 1984 Jan, 11(1), 105 - 8 A detailed physical and genetic map of two R.ColBM IncFIII plasmids; Massie B et al.; The cleavage and genetic maps of two closely related R.ColBM IncFIII plasmids, designated pSAS1201 and pSAS1203, are presented . Restriction analysis of both plasmids with SstI, EcoRI, Bg/II, XhoI, HindIII, and Sa/I indicated that the maps of these two plasmids are superimposable with the exception of a 1.70-MDa DNA segment absent in pSAS1203 . Our results provide the first physical and genetic maps of plasmids belonging to the IncFIII incompatibility group. Plasmid, 1984 Jan, 11(1), 102 - 4 Restriction enzyme maps of the chimeric R/Ent plasmid pCG86 and the related ent plasmid P307; Picken RN et al.; Complete restriction enzyme cleavage site maps for three enzymes and incomplete maps for four enzymes have been constructed for the chimeric R/Ent plasmid pCG86 of Escherichia coli and the related Ent plasmid P307. Mol Biol (Mosk), 1984 Jan-Feb, 18(1), 161 - 8 {Binding of NR1 plasmid DNA to membrane during replication in Escherichia coli mini-cells}; Perebitiuk AN et al.; Studying the replication of NR1 plasmid in E . coli mini-cells it was shown that the character of bond between plasmid DNA and membrane depends on the stage of replication cycle of the plasmid . On initiation the DNA-membrane complex is sensitive to the action of ionic force . In the process of elongation the bond of DNA molecules with the membrane is unstable if exists at all, and can be broken even by the nonionic detergent . At the final stage of replication the newly synthesized molecules form a complex with the membrane structures which is unstable in the presence of 0,5 M NaCl . The destruction of the complex followed by the open cycle of plasmid DNA coming out of it takes place under the action of ionic detergent. Mol Cell Biochem, 1984, 58(1-2), 187 - 91 Is there a general paradigm of cyclic AMP action in eukaryotes? Pall ML. The cyclic AMP control system in eukaryotes has been highly conserved evolutionarily in four of its central properties . Such conservation suggests conservation of the regulatory function of cyclic AMP . Conservation is seen in the properties of adenylate cyclase, cyclic AMP-dependent protein kinase and, among diverse lower eukaryotes, the control of endogenous cyclic AMP levels . A conserved regulatory response to cyclic AMP is the stimulation of glycolysis and inhibition of gluconeogenesis . The control of glycolysis and gluconeogenesis is proposed to be evidence of general pattern of cyclic AMP action in many lower and higher eukaryotic cells. Mol Gen Genet, 1984, 193(3), 543 - 53 Formation of type II F-primes from unstable Hfrs and their recA-independent conversion to other F-prime types; Buysse JM et al.; Four E . coli Hfr strains, representing stable (Hfr Cavalli), moderately stable (AB312) and unstable (Ra-1, Ra-2) Hfr states, were used in the isolation of a series of F' plasmids . Type II F's were found to be the most prevalent F' plasmid formed from all of the Hfrs, while the percentages of delta tra F's increased as the stability of the Hfr increased . Two observations suggested that F' formation in unstable Hfrs like Ra-2 may proceed through a type II F' precursor . First, the major F' products of Ra-2 are tra+ type II F's and, second, other F' types (I, II) and classes (tra+, delta tra) from Ra-2 appeared to be deletion derivatives of a larger F' progenitor . By monitoring the molecular changes that occur when the Ra-2 derived type II F' pWS200 is transferred from one recA host to another, we have found that all F' types and classes can be generated from pWS200 in a recA-independent manner . F sequences involved in the genetic conversions of pWS200 include the oriT locus and the directly repeated gamma delta junctions of F and chromosomal DNA . A model for the formation of F's in unstable Hfrs is postulated in which a tra+ type II F' primary excision product is seen to be modified, through recA-independent processes, to other F' types and classes . This model differs from the current model of F' formation in that independent excision events from the Hfr chromosome are not seen as the source of type I and type II F's . These studies have also shown that the formation of delta tra F's is a recA-independent process that can occur from the F' and Hfr states, that gamma delta-mediated deletions in pWS200 often demonstrate regional specificity in having endpoints near the ilv operon and that genetic alterations in either replication origin of pWS200 (F oriV, chromosomal oriC) stabilize the replication of this "mini-Hfr" cointegrate. Mol Gen Genet, 1984, 193(3), 520 - 4 Effect of the plasmid ColIb-P9 on cellular processes related to DNA repair; Kopylov VM et al.; The plasmid ColIb-P9 introduced into Escherichia coli K12 umuC mutant cells suppresses the deficiencies in mutagenesis and repair of mutants after UV-irradiation . These data suggest that ColIb-P9 encodes a product with a function similar to that of the chromosomal gene umuC . Tn5 insertion mutants of ColIb-P9 were isolated with an altered ability to restore UV-mutagenesis in the umuC mutant . The same plasmid mutations were shown to eliminate the effects of ColIb-P9 on UV-mutagenesis, survival after UV and mitomycin C treatment, reactivation of UV-irradiated lambda in unirradiated cells, Weigle-reactivation, induction of colicin E1 synthesis . The ColIb-P9 genes responsible for the enhancement of UV-mutagenesis were cloned within a 14 Md SalI fragment . Their location was established by restriction analysis of the mutant plasmid ColIb 6-13::Tn5 . While the action of the plasmids ColIb-P9 and pKM101 is similar, these plasmids were shown to have opposite effects on cell survival and colicin E1 synthesis after mitomycin C treatment . A study of the mutant plasmids ColIb::Tn5 and pGW12 (muc- mutant of pKM101) has shown the difference in the effects of ColIb-P9 and pKM101 to be associated with the plasmid genes responsible for the protective and mutagenesis-enhancing effects of these plasmids in UV-irradiated cells. Mol Gen Genet, 1984, 193(3), 513 - 9 Plasmid rescue - a tool for reproducible recovery of genes from transfected mammalian cells? Kiessling U, Platzer M, Strauss M. The efficient rescue of plasmids containing the thymidine kinase gene (tk) of Herpes simplex virus type I from genetically transformed mouse cells by transformation of bacteria is described . Rescued plasmids contain insertions of calf DNA used as a carrier in the transfection but usually lack portions of plasmid DNA . Deletions generally concern the region spanning from around the PvuII site of pBR322 to within the tetracycline resistance coding sequence, whereas the extent of tk sequence deletion varies, depending on the site of its integration (BamHI or PvuII) into the plasmid . Modelling the rescue process by transformation of bacteria with a mixture of original plasmids and sheared mouse cell DNA clearly demonstrates that deletions are caused by the presence of the mammalian DNA and they probably occur during re-transformation of bacteria before the onset of tetracycline gene expression . Plasmids lacking the Tcr region are reproducibly rescuable without deletion . Methods for reproducible re-isolation of transferred genes from mammalian cells are discussed. Mol Gen Genet, 1984, 193(3), 493 - 9 Characterization of the new insertion sequence IS91 from an alpha-hemolysin plasmid of Escherichia coli; Diaz-Aroca E et al.; IS91 is a 1.85 kb insertion sequence originally resident in the alpha-hemolytic plasmid pSU233 . The element was transposed sequentially from this plasmid to pACYC184, to R388, and to pBR322 . Both cointegrates and simple insertions of the element were obtained . A detailed restriction enzyme map of the element is presented . This does not bear any relationship to the maps of previously described insertion sequences . Furthermore, hybridization between these sequences and IS91 could not be demonstrated . Deletion derivatives of IS91 were constructed which are unable to transpose . However, their transposition can be complemented in trans by wild-type elements . One of these deletion derivatives has been genetically labeled with a kanamycin resistance marker from Tn5 . When this new element was complemented for transposition, only about 2% of the transposition products were cointegrates . Thus, the behavior of IS91 is better explained by transposition models that allow direct transposition. Mol Gen Genet, 1984, 193(3), 414 - 21 Sequence of the regulatory region of omp T, the gene specifying major outer membrane protein a (3b) of Escherichia coli K-12: implications for regulation and processing; Gordon G et al.; The DNA of the promoter region of omp T, including the putative start for the pro-Omp T protein (pro-protein a), has been sequenced . Previous studies showed that trypsin inhibitors prevent the processing of pro-Omp T to Omp T protein which led to the prediction that the processing site would be a lysine or an arginine . The deduced amino acid sequence contains a lysine at amino acid 12 and an arginine at amino acid 17 from the N terminus . Chou-Fassman analysis would predict processing at the lysine (but not the arginine) to remove a 1389 dalton peptide, consistent with the fact that the estimated molecular masses of pro-Omp T and Omp T are 42 kd and 40 kd respectively . In addition, the predicted mRNA of the promoter region can form a stable secondary structure (-17.1 kcal) that sequesters the Shine-Dalgarno (SD) sequence as well as the initiator AUG codon . There is evidence that the per A (tpo, envZ) gene product is required for synthesis of Omp T protein (as well as several outer membrane and periplasmic proteins) . The perA gene product could be activating translation of Omp T protein by disrupting the mRNA secondary structure that sequesters the SD sequence . Omp T protein synthesis is reduced at temperatures below 32 degrees C and this may also be related to the greater stability of the sequestered SD sequence of the mRNA at low temperature. EMBO J, 1984 Jan, 3(1), 87 - 9 Genes involved in transitory recombination between phage M13 and plasmid pHV33; Dagert M et al.; Plasmid pHV33 and phage M13 combine in Escherichia coli cells to form a chimera, which decombines to regenerate two parental genomes . Combination can occur via two genetic pathways, one defined by the recBC genes, the other by recA, recF and possibly recL genes . Decombination can also occur via two pathways, one defined again by the recBC genes, the other by a gene not identified, but active only in the absence of the recL gene product. EMBO J, 1984 Jan, 3(1), 43 - 50 On the action of the cyclic AMP-cyclic AMP receptor protein complex at the Escherichia coli lactose and galactose promoter regions; Spassky A et al.; Using DNase footprinting and transcription assays in vitro we have probed the effect of the cAMP-cAMP receptor protein complex (cAMP-CRP) on the positioning of RNA polymerase and on the location of the transcription start point at the Escherichia coli gal and lac operon regulatory regions . In both cases, RNA polymerase can form two alternative complexes which promote transcription from two different start points, S1 and S2: pre-incubation of promoter DNA with cAMP-CRP results in a shift of the transcription start from S2 to S1 and in an increase in the rate of open complex formation . Moreover, the rate of formation of each heparin-resistant complex parallels the establishment of the corresponding footprint, showing that the stable binding corresponds to open complex formation . We show that, in the case of gal, RNA polymerase, which is bound so as to transcribe from S2, cannot be diverted to S1 by subsequent addition of cAMP-CRP . In contrast, in the case of lac, when cAMP-CRP is added after RNA polymerase, complexes which initiate transcription at S2 are rapidly converted to complexes which initiate at S1 . Finally, we present data which suggest that protein-protein interactions are essential for CRP-induced activation at both the lac and gal promoters. EMBO J, 1984 Jan, 3(1), 179 - 83 Structure and function of the intercistronic regulatory deoC-deoA element of Escherichia coli K-12; Valentin-Hansen P et al.; The deoC-deoA intercistronic region from Escherichia coli has been characterized by DNA and protein sequencing . This region consists of 129 bp and includes a regulatory element, which has also been found in other operons containing large intercistronic regions . Our results suggest that the regulatory element functions as a transcriptional attenuator, and therefore it seems likely that the primary role of this element is to effect differential expression of operon genes . Furthermore, we have unambiguously shown that the initiation codon for the deoA gene is UUG. Appl Environ Microbiol, 1984 Jan, 47(1), 15 - 21 Cloning and sequencing of the xylose isomerase and xylulose kinase genes of Escherichia coli; Lawlis VB et al.; A 4.2-kilobase-pair fragment of the Escherichia coli chromosome which contains the genes for xylose isomerase and xylulose kinase was cloned into plasmid pBR322 . The hybrid plasmid (designated pECX14) complements strains deficient in either or both of the two enzymes . Deletion derivatives of pECX14 were used to localize the two genes on the cloned fragment . The entire nucleotide sequence of the cloned fragment was determined . Open reading frames which, if translated, would encode proteins of molecular weights 54,000 and 52,000 were found . These were identified as the isomerase and kinase structural genes, respectively. Proc Natl Acad Sci U S A, 1984 Jan, 81(2), 550 - 4 Escherichia coli mutants suppressing replication-defective mutations of the ColE1 plasmid; Naito S et al.; Mutants of Escherichia coli K-12 have been isolated that suppress cer mutants, ColE1 mutants that are unable to replicate as the plasmid . These host suppressors were designated her, for host factor affecting ColE1 replication . Each her suppressor showed a characteristic pattern of suppression depending on the cer mutation used for selecting the mutant bacteria . One of the suppressors, named herA, that suppressed cer6, a single-base-pair alteration 160 base pairs upstream of the ColE1 replication origin, was genetically identified as an alteration of the rnh gene (RNase H) . HerA was recessive to its wild-type allele . RNase H activity of herA cell extracts was defective . Conversely, rnh mutants that were isolated independently of ColE1 replication supported replication of cer6 DNA . Some rnh mutants manifested the HerA phenotype only above a certain transition temperature, and their RNase H activity was found to be temperature sensitive . Therefore, replication of cer6 DNA in vivo is sensitive to RNase H activity . Under the conditions that suppressed cer6, the wild-type colE1 replicon replicated normally . Then, ColE1 replication in vivo proceeds in the absence of RNase H activity, which has been shown to be required for in vitro replication of the DNA. Proc Natl Acad Sci U S A, 1984 Jan, 81(2), 503 - 7 Correction of complex heteroduplexes made of mouse H-2 gene sequences in Escherichia coli K-12; Cami B et al.; We have prepared heteroduplexes between two plasmids that carry, in the same orientation, two H-2 cDNA inserts, 1.15 and 1.0 kilobase long, respectively . Their sequences encode two distinct class I transplantation antigens of the mouse and differ by 8% of their nucleotides . Molecules with a rearranged array of restriction sites were found after transformation and cloning in an Escherichia coli recA- host . Nucleotide sequences showed that the rearranged molecules derived their nucleotides from the two parental strands . Thus, correction of these complex heteroduplexes takes place in E . coli and probably involves repair mechanisms . It provides the basis for a mutational process in which several nucleotides (amino acids) can be altered in a single event . It also offers a practical means of making genetic variants . Several other implications are discussed. Proc Natl Acad Sci U S A, 1984 Jan, 81(1), 3 - 7 Secondary structure specificity of the nuclease activity of the 1,10-phenanthroline-copper complex; Pope LE et al.; The artificial DNase activity of the 1,10-phenanthroline-cuprous ion complex {(OP)2Cu+} and H2O2 cleaves the A, B, and Z forms of DNA at different rates . The B structure, formed by most DNAs including poly(dA-dT) and poly(dA) X poly(dT), is the most susceptible to cleavage . It is completely degraded within 1 min by 40 microM 1,10-phenanthroline/4 microM Cu2+/7 mM H2O2/7 mM 3-mercaptopropionic acid . The A structure, formed by RNA X DNA hybrids such as poly(rA) X poly(dT), is cleaved in both strands roughly 10-20% as rapidly as poly(dA-dT) under comparable conditions . In contrast, the left-handed Z structure, formed by poly(dG-dC) in 3.0 M NaCl, is completely resistant to cleavage even though the same copolymer in the B structure at 15 mM NaCl is readily degraded . Poly(dA-dT) is rendered acid soluble at both salt concentrations at similar rates . The basis for the secondary structure specificity of the DNA cleavage reaction most likely resides in the requisite formation of a productive complex between (OP)2Cu+ and DNA during the course of this reaction . Previous studies have suggested that strand scission is due to oxidative destruction of the deoxyribose by hydroxyl radicals produced by the oxidation of DNA-bound Cu+ by H2O2 . Apparently, the Z and A structures are unable to form a stable noncovalent complex with the same optimal geometry for cleavage as the B structure and are less susceptible to degradation . This artificial DNase activity may provide an approach to assess the formation of non-B-DNA structures in solution. Proc Natl Acad Sci U S A, 1984 Jan, 81(1), 13 - 7 Construction and characterization of extrachromosomal probes for mutagenesis by carcinogens: site-specific incorporation of O6-methylguanine into viral and plasmid genomes; Green CL et al.; Organic synthesis and recombinant DNA technology were used to situate a putatively premutagenic DNA lesion, O6-methylguanine (O6MeGua), at a specific location in the genomes of two bacterial viruses, M13mp8 and phi X174, and of the bacterial plasmid pBR322 . In each genome the first guanine residue in the unique recognition sequence for restriction endonuclease Pst I (5'-C-T-G-C-A-G-3') was replaced with O6MeGua . This was accomplished by ligating a chemically synthesized tetranucleotide, 5'-pTpm6GpCpA-3', into a circular, genome-length heteroduplex in which the four internal nucleotides of the Pst I recognition site had been removed from one strand of the DNA double helix (ligation yield, approximately equal to 50%) . It was established that the tetranucleotide was located specifically at the Pst I site and that the presence of O6MeGua rendered the ligation product resistant to cleavage by Pst I . Sensitivity of the genome to Pst I was restored upon treatment with purified Escherichia coli O6MeGua DNA-methyltransferase, a repair protein that removes the methyl group from DNA-bound O6MeGua . This result, in combination with other data, showed unambiguously that O6MeGua was incorporated with high yield into the Pst I recognition sequence. Mol Gen Genet, 1984, 193(2), 376 - 8 Characterization of a glpK transducing phage; Conrad CA et al.; A specialized glpK transducing phage, lambda glpK100, has been isolated and characterized with respect to DNA structure . The glpK component of the glpKF operon has been localized within a 2.0 kilobase pair (kbp) region of the approximately 8.24 kbp bacterial DNA insert, and the positions of BamHI, EcoRI and HindIII restriction sites within this DNA have been identified. Mol Gen Genet, 1984, 193(2), 370 - 5 Mutagenesis of the metJBLF gene cluster with transposon Tn5: localization of the metF transcription unit; Treat ML et al.; Mutants of a specialized lambda dmet transducing phage bearing the metJBLF gene cluster of Escherichia coli K12 were constructed using transposon Tn5 . Two of these mutants, lambda dmet128::Tn5, MW77 and lambda dmet128::Tn5, 3-1, were used to locate precisely as well as confirm the existence of the metF transcription unit (approximately 1,000 base pairs in size) . The introduction of new restriction sites within the metJBLF gene cluster due to the Tn5 insertion events allowed the metF transcription unit to be cloned into the high copy number plasmid pBR322 . Analyses of the structures of two of these recombinant plasmids, pTJ77H and pTJ13-1H, are presented . Expression of the plasmid borne metF allele in cells grown in the absence, or presence, of exogenous L methionine (0.2 mM) demonstrates that the amplification of the metF copy number does not abolish met regulon mediated control of the gene's activity. Mol Gen Genet, 1984, 193(2), 349 - 57 Genetic structure and stability of a copy number mutant of IncFI group plasmid ColV-K94 in Escherichia coli K-12; Mitra G et al.; Mutants pWS10, pWS11 and pWS12 were derived from an IncFI group plasmid ColV-K94 by the insertion of a transposon Tn903 (Kmr) . These plasmids were all approximately 130 kb in length . The plasmid pWS12 resembled the wild type ColV-K94 in transmissibility, incompatibility and stable maintenance . Cells harboring pWS11 were poor conjugal donors but resistant to the same level of kanamycin as pWS12 containing hosts . In contrast, pWS10 conferred a higher resistance to kanamycin and exhibited reduced incompatibility properties in comparison with pWS12 . The higher drug resistance associated with pWS10 appeared to be a consequence of an increase in its copy number and the generation of miniplasmids of varying sizes . Electron microscope analysis of the copy mutant pWS10 revealed that Tn903 was located at a site adjacent to a region 32.6F to 35.3F . The latter region appears to be the primary replicon of ColV-K94 and is homologous with the secondary replicon of F . The insertional mutagenesis with Tn903 brought about an extensive DNA rearrangement including the duplication and translocation of the stems of two inverted repeat structures . The DNA alterations of pWS10 were distinguishable through comparison of its EcoRI digestion patterns with those of pWS11 and pWS12. Mol Gen Genet, 1984, 193(2), 340 - 8 Benzyl derivative facilitation of transcription in Escherichia coli at the ara and lac operon promoters: metabolite gene regulation (MGR); Kline EL et al.; A number of benzyl derivatives have been tested for their ability to induce the expression of the araBAD operon in an Escherichia coli K-12 strain . Those derivatives shown to be stimulatory include: benzoic acid (BA), para-amino benzoic acid (PABA), para-hydroxy benzoic acid (PHBA), ortho-amino benzoic acid (OABA), 3-hydroxy-4-methoxy phenylethylamine (MTA), and 4-hydroxy-3-methoxyphenol acetic acid (HVA) . The araC gene product was necessary to facilitate the induction . To further characterize if the inductive effect was mediated at the level of transcription, an araBAD-tetracycline resistant (Tcr) operon fusion plasmid (pAP-B) was employed . Benzyl derivatives which induce expression of the araBAD operon in situ also induced a Tcr phenotype with pAP-B . Both indole acetic acid (IAA) and imidazole (IM), which were previously shown to circumvent the necessity for cAMP in the induction of the araBAD operon, also induced a Tcr phenotype with pAP-B . Induction of lac or other cAMP responding operons with the inducing molecules at the chromosomal level was not detectable when assessed by carbon utilization . However, a lacZYA-Tcr operon fusion plasmid (pLPI) did respond to IAA and several of the inducing benzyl derivatives . Catabolite repression of chromosomal araBAD expression was reversed when the exogenous concentration of OABA was elevated . Similar effects on the Tcr phenotypes conferred by pAP-B and pLP1 were observed when OABA or several other inducing benzyl derivatives were present exogenously. Mol Gen Genet, 1984, 193(2), 316 - 21 UV inducible UV protection and mutation functions on the I group plasmid TP110; Dowden SB et al.; The UV protection and mutation properties of the I group plasmid TP110 have been investigated . It is demonstrated that the genes responsible for these effects are able to complement the deficiency in umuC36 mutants of E . coli, as are the similar genes carried by the B group plasmid R16 . Mu-lac inserts into TP110 have been isolated which abolish the UV protection and mutation functions . Restriction mapping of these inserts locates them within a single region of the genome . A comparison of the restriction sites of this region with the muc region of pKM101 reveals very little similarity . Expression of beta-galactosidase in those Mu-lac inserts in which the lacZ gene is fused to the promoter for the protection and mutation functions is inducible by DNA damaging agents, and induction in mutant strains suggests that these genes are under the direct control of the lexA repressor. Mol Gen Genet, 1984, 193(2), 269 - 74 Enhanced recombination between lambda plac5 and mini-F-lac: the tra regulon is required for recombination enhancement; Seifert HS et al.; F42lac recombination with lambda plac is normally 20-fold to 50-fold higher than recombination between lambda plac and a chromosomal lac gene . Transductional crosses with lambda plac and a recombinant plasmid containing the lac operon and F replication functions show only two to four-fold higher recombination than similar crosses with a chromosomal lac gene . Insertion of a BamHI fragment containing the entire tra regulon of F into the mini-F-lac plasmid restores the high level of recombination seen with F42lac. Mol Gen Genet, 1984, 193(2), 263 - 8 Deletion of 60 kilobase pairs of DNA from the terC region of the chromosome of Escherichia coli; Henson JM et al.; The terminus of replication (terC) of the chromosome of Escherichia coli is located between the rac (min 30.0) and manA (min 35.7) loci, presumably close to the trg (min 31.4) locus . We have used a strain containing lambda reverse (min 30.0) and trg-2::Tn10 (min 31.4) to obtain deletions of the entire 60 kilobase pair region that separates these elements . Strains harboring these deletions possessed fusion fragments that contained DNA homologous to both lambda reverse and trg region DNA . In addition, chromosomal DNA normally present between min 30.0 and min 31.4 was absent in these strains . The strains had no readily apparent mutant phenotype, which demonstrates that this large region of DNA is not essential for normal growth. Mol Gen Genet, 1984, 193(2), 244 - 50 Transcription of the trpR gene of Escherichia coli: an autogeneously regulated system studied by direct measurements of mRNA levels in vivo; Bogosian G et al.; The expression of the trpR gene of Escherichia coli was investigated by measuring trpR messenger RNA levels in vivo under various physiological conditions . Trp repressor, when present, led to significant decreases in the amount of trpR message produced; this effect was enhanced by providing excess L-tryptophan to the system . In the absence of Trp repressor, no changes in trpR message levels were observed under any of the conditions employed . Sedimentation profiles of trpR mRNA revealed a single species under all circumstances . These results suggest that autogenous repression alone acts to regulate transcription of the trpR gene . The activity of the trpR promoter in vivo was evaluated using a trpR-lacZ operon fusion . Very good agreement was found between relative promoter activity and trpR message levels under all experimental conditions. Mol Gen Genet, 1984, 193(2), 238 - 43 Homology is not required for recombination mediated by DNA gyrase of Escherichia coli; Naito A et al.; We have previously shown that DNA gyrase of Escherichia coli can promote recombination between heterologous DNAs in a cell-free system (Ikeda et al . 1982) . In the present paper, we have studied the nucleotide sequences of several recombination junctions of lambda-pBR322 recombinants and found that there were not more than three-basepair homologies between the parental DNAs in six combinations of the lambda and pBR322 recombination sites . Based on this and previous results, we concluded that homology was not required for the DNA gyrase-mediated recombination . Furthermore, the structures of the recombinant DNAs we have analyzed suggest the occurrence of multiple cross-overs in our in vitro system. Mol Gen Genet, 1984, 193(2), 231 - 7 Function of RNase H in DNA replication revealed by RNase H defective mutants of Escherichia coli; Ogawa T et al.; Escherichia coli rnh mutants were isolated using localized mutagenesis and selective measurements of RNase H activity in mutagenized cell extracts with {3H}poly(rC) X poly(dG) as substrate . RNase H activity in extracts of one mutant, ON152 (rnh-91), was undetectable (less than 0.05% of that of wild-type cells) . This mutant formed small colonies at 43 degrees C . At this temperature, accumulation of nascent fragments was more prominent in the rnh-91 X polA4113 double mutant than in the polA4113 mutant; however, no accumulation was found in the rnh single mutant at 43 degrees C . Unlike the 1-3 nucleotide primer RNA found on nascent fragments of polA4113 cells, primers from the rnh-91 X polA4113 cells ranged from one to about ten bases . These results suggest that the 5' leads to 3' exonuclease activity of DNA polymerase I plays a major role in removal of primer RNA and that RNase H functions in an auxiliary role, excising the 5'-portion of longer primers . The rnh mutant supports replication of ColE1-type plasmids . A possible mechanism of replication of such plasmids in rnh mutants and a role of RNase H in the initiation of chromosomal replication are discussed. Mol Gen Genet, 1984, 193(2), 225 - 30 Cloning of the dnaB gene of Escherichia coli: the dnaB gene of groPB534 and groPB612 and the replication of phage lambda; Gunther E et al.; Fragments of the E . coli chromosome that carry the dnaB groPB534 or groPB612 alleles have been cloned into a cosmid vector . The resulting recombinant plasmids contained the genes uvrA, groP (B534 or B612), and lexA . Further subcloning into high copy number plasmids, during which the uvrA and lexA genes were removed successively, yielded a groPB534 and groPB612 DNA fragment of about 2.4 kb each . Both fragments contained an overlapping 1.8 kb segment of DNA in which the sites of all restriction enzymes tested were identical . The size of these dnaB gene fragments were further delimited by deletion analysis . In E . coli groPB534 in which lambda wild-type and lambda pi A mutants do not replicate (Georgopoulos and Herskowitz 1971) phage replication is rescued if the strain contains the groPB534 gene on high copy number plasmids . On the contrary, in E . coli groPB612, which is temperature-sensitive for its groP character, replication of lambda and lambda pi A is abolished at 30 degrees C if the strain contains the groPB612 recombinant plasmid . On the other hand, replication of lambda pi B remains unaffected whether or not the groP strains harbor the isogenic dnaB gene-containing plasmid . The results suggest that within the cell not only the quality but also the relative amounts of dnaB and lambda P protein are crucial for lambda phage replication. Biull Eksp Biol Med, 1984 Jan, 97(1), 86 - 8 {Formation of cointegrates during mobilization of nonconjugated plasmids to genetic transfer by plasmid pAP42}; Khamidullina RG et al.; pAP42 plasmid mobilizes nonconjugative pMR5, pBR322, pACYC 184, RSF2124, RSF1010 plasmids with different frequency . During mobilization of nonconjugative RSF2124 and pACYC184 plasmids by pAP42 factor there form cointegrative structures similar in their properties to typical R plasmids. Mol Gen Genet, 1984, 193(1), 72 - 5 Construction and analysis of plasmids containing the Escherichia coli serB gene; Garnant MK et al.; Plasmid pserB59-1 carries the E . coli serB gene on a 5.2 kb BamHI fragment cloned into the BamHI site of plasmid pBR322 . The results of genetic and biochemical experiments established that a functional serB gene is contained in the fragment . The location of the serB gene within the insert was determined by restriction endonuclease analysis of plasmids derived from pserB59-1 that carry the Tn5 element at sites that inactivate the serB gene, and by deletions of segments of the 5.2 kb insert that either inactivate or do not inactivate the serB gene . A 38,000 Mr serB+ polypeptide was detected when plasmid pserB59-1 was used as template in a minicell system, but not when the serB gene was inactivated by insertion of a Tn5 element. Mol Gen Genet, 1984, 193(1), 64 - 71 The promoters of the atp operon of Escherichia coli K12; Nielsen J et al.; The nucleotide sequence has been determined of a 900 bp segment of chromosomal DNA located between 2.6 and 3.5 kb left of the origin of replication, oriC . This segment, which overlaps with the known sequence of the atp operon coding for the eight subunits of the Escherichia coli K12 ATP synthase, contains two coding sequences with the same polarity (counterclockwise) as the atp genes: One of these, designated atpI, which codes for the N-terminal part of a 14 kD polypeptide, is located in front (upstream) of the atpB gene (the first structural gene in the atp operon), the other one codes for the C-terminal part of the gidB gene . The 606 bp segment located between the gidB and the atpI genes contains no coding sequences . By employing the nuclease S1 mapping technique, we have determined a promoter, designated atpIp, for the atp operon located in front of the atpI gene; two additional, weak transcription starts were located within the atpI gene . No transcription start sites were detected up to 1,000 bp upstream of the atpIp promoter, neither were any transcription start sites detected within the cluster of the eight structural atp genes . The atp operon transcription terminates at a site approximately 50 bp downstream from the atpC gene. Mol Gen Genet, 1984, 193(1), 179 - 87 Genes for gentamicin-(3)-N-acetyltransferases III and IV: I . Nucleotide sequence of the AAC(3)-IV gene and possible involvement of an IS140 element in its expression; Brau B et al.; Genes for gentamicin-3-acetyltransferases {ACC(3)} of types III and IV have been cloned from various R-plasmids . In two R-plasmids, pWP14a (AAC(3)-III) and pWP7b {AAC(3)-IV}, resistance genes have been found directly adjacent to a single copy of an IS element, IS140 . Nucleotide sequence determination of the AAC(3)-IV gene from plasmid pWP7b and of part of IS140 from three different sources suggested that the -35 region of the AAC(3)-IV promoter was part of the IS element . A similarly built-up promoter was found in pWP14a . It was found also, that a hygromycin B phosphotransferase was expressed from a locus neighbouring the AAC(3)-IV gene in pWP7b which was under the control of the same promoter . In two other R-plasmids, pWP113a and pWP116a, the AAC(3)-III gene was found in different genetic environments, namely close to Tn3-like structures. Mol Gen Genet, 1984, 193(1), 162 - 6 Resolution of a hybrid cointegrate between transposons Tn501 and Tn1721 defines the recombination site; Rogowsky P et al.; The related transposons Tn501 and Tn1721 have a 3.8 kb region in common that contains two genes (tnpA and tnpR) and a resolution site (res) required for transposition . Resolvase, the product of tnpR, catalyses site-specific recombination at res, a 186 base pair (bp) sequence located adjacent to tnpR at one end of the homology region . We describe here identification of the crossover site within res . It involved the construction of a plasmid containing copies of res (Tn501) and res (Tn1721) in direct orientation and tnpR-mediated intramolecular recombination between the two homologous (but non-identical) sites . The resulting hybrid contained Tn501 and Tn1721 fused at the crossover point . DNA sequence analysis of the recombinant indicates that recombination occurs in an 11 bp region of exact homology between Tn501 and Tn1721 . The recombination site lies 161-172 bp upstream of tnpR at the transition from homology to non-homology between Tn501 and Tn1721 suggesting that site-specific recombination may have played a role in the evolution of these elements. Mol Gen Genet, 1984, 193(1), 110 - 8 Analysis in vivo of factors affecting the control of transcription initiation at promoters containing target sites for trp repressor; Bogosian G et al.; An investigation of repression in the trp system of Escherichia coli was undertaken using operon fusions and plasmids constructed via recombinant DNA technology . The promoters of the trp operon and the trpR gene were fused to lacZ, enabling the activity of these promoters to be evaluated under various conditions through measurements of beta-galactosidase production . In confirmation of earlier studies, the trpR gene was shown to be regulated autogenously . This control feature of the trp system was found to maintain intracellular Trp repressor protein at essentially invariant levels under most conditions studied . Increasing the trpR+ gene dosage did not significantly elevate Trp repressor protein levels, nor did the introduction of additional operator "sinks" result in significantly decreased levels of Trp repressor protein . Definite alterations in intracellular Trp repressor protein levels were achieved only by subverting the normal trpR regulatory elements . The placement of the lacUV5 or the lambda PL promoters upstream of the trpR gene resulted in significant increases in repression of the trp system . Substituting the primary trp promoter/operator for the native trpR promoter/operator resulted in an altered regulatory response of the trp system to tryptophan limitation or excess . The regulation of the trpR gene effectively imparts a broad range of expression to the trp operon in a manner finely attuned to fluctuations in intracellular tryptophan levels. J Virol, 1984 Jan, 49(1), 236 - 47 Fine mapping and molecular cloning of mutations in the herpes simplex virus DNA polymerase locus; Coen DM et al.; Mutations in five phenotypically distinct mutants derived from herpes simplex virus type 1 strain KOS which lie in or near the herpes simplex virus DNA polymerase (pol) locus have been fine mapped with the aid of cloned fragments of mutant and wild-type viral DNAs to distinct restriction fragments of 1.1 kilobase pairs (kbp) or less . DNA sequences containing a mutation or mutations conferring resistance to the antiviral drugs phosphonoacetic acid, acyclovir, and arabinosyladenine of pol mutant PAAr5 have been cloned as a 27-kbp Bg+II fragment in Escherichia coli . These drug resistance markers have been mapped more finely in marker transfer experiments to a 1.1-kbp fragment (coordinates 0.427 to 0.434) . In intratypic marker rescue experiments, temperature-sensitive (ts), phosphonoacetic acid resistance, and acyclovir resistance markers of pol mutant tsD9 were mapped to a 0.8-kbp fragment at the left end of the EcoRI M fragment (coordinates 0.422 to 0.427) . The ts mutation of pol mutant tsC4 maps within a 0.3-kbp sequence (coordinates 0.420 to 0.422), whereas that of tsC7 lies within the 1.1-kbp fragment immediately to the left (coordinates 0.413 to 0.420) . tsC4 displays the novel phenotype of hypersensitivity to phosphonoacetic acid; however, the phosphonoacetic acid hypersensitivity phenotype is almost certainly not due to the mutation(s) conferring temperature sensitivity . The ts mutation of mutant tsN20--which does not affect DNA polymerase activity--maps to a 0.5-kbp fragment at the right-hand end of the EcoRI M fragment (coordinates 0.445 to 0.448) . The mapping of the mutations in these five mutants further defines the limits of the pol locus and separates mutations differentially affecting catalytic functions of the polymerase. J Bacteriol, 1984 Jan, 157(1), 314 - 7 Shuttle vectors for cloning recombinant DNA in Escherichia coli and Streptomyces griseofuscus C581; Larson JL et al.; The replicon of the Streptomyces plasmid SCP2 was located on a 5.9-kilobase EcoRI-SalI restriction fragment . The SCP2 replicon was combined with Escherichia coli plasmid pBR322 and genes specifying neomycin resistance and thiostrepton resistance in streptomycetes to construct shuttle vectors that are useful for cloning in E . coli and streptomycetes. J Bacteriol, 1984 Jan, 157(1), 303 - 10 Isolation and analysis of aroFo mutants by using an aroF-lac operon fusion; Cobbett CS et al.; The lac structural genes were fused to the regulatory region of the aroF-tyrA operon so that the expression of beta-galactosidase was regulated by the tyrR+ gene product . Transducing phage carrying the aroF-lac fusion were isolated, and a lambda aroF-lac lysogen was used to select for aroFo mutants . A plasmid vector was constructed onto which the aroFo mutations were transferred by recombination in vivo. J Bacteriol, 1984 Jan, 157(1), 262 - 8 Gene envY of Escherichia coli K-12 affects thermoregulation of major porin expression; Lundrigan MD et al.; The temperature-dependent expression of OmpF and OmpC, the major channel-forming proteins of the Escherichia coli K-12 outer membrane, was studied . In wild-type cells, decreasing growth temperatures resulted in increased amounts of OmpF protein and correspondingly decreased quantities of OmpC protein . Bacteria deleted for the 13-min chromosomal region did not exhibit this temp