Mol Biol (Mosk), 1985 Mar-Apr, 19(2), 545 - 52 {Study of the photoaffinity modification of Escherichia coli ribosomes near the donor tRNA-binding center}; Bausk EV et al.; Affinity labelling of E . coli ribosomes near the donor tRNA-binding (P) site was studied with the use of photoreactive derivatives of tRNAPhe bearing arylazidogroups on N7 atoms of guanine residues (azido-tRNA) . UV-irradiation of complexes 70S ribosome.poly(U).azido- tRNA(P-site) and 70S ribosome.poly(U).azido-tRNA(P-site).Phe- tRNAPhe(A-site) resulted in covalent attachment of azido-tRNA to ribosomes, both subunits being labelled . In both cases modification extent of 30S subunit was two-fold than that of the 50S one . It was shown that when the A-site was free the azido-tRNA located in P-site labelled proteins S9, S11, S12, S13, S21 and L14, L27, L31 . Azido-tRNA located in P-site when the A-site was occupied with Phe-tRNAPhe labelled proteins S11, S12, S13, S14, S19, L32/L33 and possibly L23, L25 . From the comparison of the sets of proteins labelled when A-site was free or occupied a conclusion was drawn that aminoacyl-tRNA located in ribosomal A-site affects the arrangement of deacylated tRNA in P-site . Data obtained allow to propose that proteins S5, S19, S20 and L24, L33 interact with guanine residues important for the tRNA tertiary structure formation. Mol Biol (Mosk), 1985 Mar-Apr, 19(2), 537 - 44 {Circular transcription map of the plasmid pBR322}; Denisova LIa et al.; The plasmid pBR322 transcription in the isolated E . coli DNA-dependent RNA-polymerase system was studied . Transcription regions as well as transcripts orientation were defined using both the technique of "criss-cross" hybridization and the annealing of RNA-products with L- and H-strands of plasmid . Summing up obtained data together with available data on promoter localization a circular transcription map of plasmid pBR322 was constructed . Effects of heparin, ion strength and E . coli S-30 system on in vitro transcripts were also studied . The 110-long RNA transcript synthesized in Ori region of pBR322 was found to be the most sensitive to all these factors . RNA-transcripts obtained in in vitro system are able to direct protein synthesis in cell-free S-30 system. Mol Biol (Mosk), 1985 Mar-Apr, 19(2), 524 - 36 {Context analysis of polynucleotide sequences . Methods of detecting non-random repeats . I . Direct repeats in genes of beta-, beta'-, sigma subunits of Escherichia coli RNA-polymerase}; Solov'ev VV et al.; A new method of contextual analysis of polynucleotide sequences in developed . The method finds nonrandom repeats in the sequences of N bases in length with given nucleotide frequencies . The coding regions of genes specifying beta-, beta'-, sigma-subunits of E . coli RNA polymerase were analyzed . The high content of short repeats was found to correspond to the secondary structure of globular proteins coded by the genes . The possible evolutionary role of the nonrandom direct repeats in coding regions of genes is discussed. Mol Biol (Mosk), 1985 Mar-Apr, 19(2), 516 - 23 {Interaction of Escherichia coli RNA-polymerase with DNA-duplexes containing repetitive sequences}; Koroleva ON et al.; Nitrocellulose filter binding technique has been used to study the binding of E . coli RNA polymerase to synthetic DNA duplexes (100-200 base pairs), containing the repeating fragments of promoters . It has been shown, that the duplex, containing the repeats of "ideal" . Pribnow box forms heparin resistant complexes with enzyme, the stability of which is comparable with that of lacUV5 promoter complexes (the half life is approximately 200 min) . The synthetic polynucleotide with repeating trp-promoter-operator sequence less stable complexes with RNA polymerase, the half life of which being 30 min. Am J Vet Res, 1985 Mar, 46(3), 591 - 6 Flunixin meglumine attenuation of endotoxin-induced damage to the cardiopulmonary vascular endothelium of the pony; Turek JJ et al.; Endotoxic shock was induced in 5 ponies by intraperitoneal injections of 20, 40, 60, 80, and 80 micrograms of Escherichia coli endotoxin (LPS)/kg of body weight at 0, 6, 12, 18, and 24 hours, respectively . At 24 hours, the ponies also were given 20 micrograms of LPS/kg via catheter in the left ventricle of the heart . A 2nd group of 4 ponies was given 1.1 mg of flunixin meglumine (FM)/kg, IV, at 6, 12, 18, and 24 hours just before the corresponding LPS injection . Two hours after the 24-hour LPS injection, the ponies in both groups were anesthetized, the lungs were perfused with fixative, and portions of the pulmonary arteries and veins and right and left ventricles were prepared for scanning and transmission electron microscopy . In ponies that were given only LPS, some areas of pulmonary vascular endothelium appeared normal when compared with untreated controls, but other areas had disoriented endothelial cells or had varying amounts of sloughing, which ranged from focal areas of a few cells to large areas of denuded endothelium . Ponies treated with FM before LPS had less severe and less extensive endothelial cell damage . In both groups, leukocytes were attached to areas of the vessel wall; endothelial cell damage was greater in these regions . Administration of FM before LPS administration attenuated the LPS-induced endothelial cell damage. Mol Cell Biol, 1985 Mar, 5(3), 448 - 56 Expression and characterization of the human c-myc DNA-binding protein; Watt RA et al.; In an effort to study in detail the nature of the protein product of the human protooncogene c-myc, we have expressed the gene at high levels in Escherichia coli . The c-myc coding region was taken from a full-length cDNA clone and inserted into a vector designed to express foreign gene products efficiently in E . coli . Pulse-labeling experiments indicated that the rate of expression of c-myc in this thermoinducible expression system is very efficient . The product was relatively stable and accumulated to approximately 10% of total cellular protein . A purification protocol was devised which allowed the c-myc protein to be readily purified in quantities sufficient for detailed biochemical and physical analyses . A high-titer polyclonal antiserum was raised against the pure protein and shown to immunoprecipitate the p110gag-myc fusion protein of MC-29-infected quail cells . This antiserum also selectively detects a protein with an apparent molecular weight of 64,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis from a Burkitt lymphoma cell line . We conclude that this 64-kilodalton protein is the human c-myc gene product since the E . coli-made protein exhibits an equivalent molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, even though its calculated molecular weight is 49,000 . Furthermore, we demonstrate that the bacterially made human c-myc protein is a DNA-binding protein and that it exhibits a high nonspecific affinity for double-stranded DNA. Invest Radiol, 1985 Mar-Apr, 20(2), 152 - 8 Computed tomographic evaluation of an experimental model for pyogenic liver abscesses; Thompson WM et al.; Computed tomography (CT) was used to evaluate 15 rabbits with experimentally induced liver abscesses . The animals were examined both before and after intravenous contrast injection . After sacrificing the animals, postfreeze CT scans were made to mark the abdomen for 1-cm thick whole body sections for correlating the gross pathology with the results of the CT scans . CT detected 15 abscesses in 13 of the 14 rabbits with true positive lesions . Ten abscesses less than 1.4 cm in diameter were not detected by CT . Contrast agent enhancement was helpful in 70% of the studies . These abscesses have characteristics similar to human liver abscesses, but there was more gas and calcium in the experimentally induced abscesses than is encountered in humans with hepatic abscesses . The model and its CT characteristics appear well suited for future studies in the diagnosis and treatment of liver abscesses. Genetika, 1985 Mar, 21(3), 384 - 90 {Escherichia coli K-12 mutants with increased resistance to ionizing radiation . V . The effect of mutations on spontaneous and radiation mutagenesis}; Bresler SE et al.; The frequencies of spontaneous mutations (reversions his-4----His+ and forward mutations to rifampicin-, nalidixic acid- or valine-resistance) in radiation-resistant mutants Gamr444 and Gamr445 are much lower than in the wild-type strain AB1157 . His+ revertants and rifampicin-resistant mutants Rifr are induced by low doses of gamma-rays more efficiently than in the wild-type . Low doses of UV light only enhanced mutagenic activity in Gamr strains for induction of His+ reversions but not for Rifr mutations . For the wild-type strain the frequencies of His+ and Rifr mutations increase proportionally to the square of dose both of UV light and gamma-rays . For the most radioresistant Gamr444 mutant the frequencies of UV- and gamma-rays-induced Rifr mutations and of gamma-rays-induced reversions increase linearly with the dose . Possible reasons for these anomalies of radiation-induced mutagenesis in Gamr mutants are discussed. Biofizika, 1985 Mar-Apr, 30(2), 366 - 71 {Is the coherence of low-intensity laser light essential for its effect on biological objects?}; Lobko VV et al.; Low-intensity laser light coherence is considered in relation to biological objects under normal physiological conditions . Estimations show that the excitation rate (the rate of coherent states generation) of typical biomolecules in visible range (sigma abs = 10(-17) cm2, I = 10(-3) W/cm2) is 10(12)-10(13) times lower than that of their phase relaxation . It means that the role of coherent interaction processes is negligible . This conclusion is confirmed by the experimental results obtained with living cells of different types. Somat Cell Mol Genet, 1985 Mar, 11(2), 177 - 87 Integration of a dominant selectable marker into human chromosomes and transfer of marked chromosomes to mouse cells by microcell fusion; Athwal RS et al.; A method for the production of stable mouse-human cell hybrids containing a single human chromosome is described . As a first step in this method, a cloned selectable marker, the E . coli xanthine-guanine phosphoribosyltransferase (Ecogpt) gene, was transferred to human cells to generate cell lines each carrying Ecogpt integrated into a different site . Human chromosomes marked with Ecogpt were transferred further into mouse cells by microcell fusion . Monochromosomal hybrids, in which the human chromosome is maintained by selection, have been produced for chromosomes 2, 5, 16, and a rearranged chromosome involving a translocation between chromosomes 1 and 2 . In addition to these monochromosomal hybrids, we have also obtained monochromosomal hybrids for human chromosomes 6, 12, and 17 by selection for the loss of marked chromosome from the microcell hybrids each containing two human chromosomes . Although the human chromosome present in these hybrids cannot be maintained by selection, 80-90% of cells retained the transferred chromosome on continuous growth for 15 days . Monochromosomal hybrids would provide biological materials to construct genetic maps of human chromosomes . In addition, chromosomes marked with dominant selectable markers can be transferred further to any cell line of interest in inter- or intra-species combination. J Surg Res, 1985 Mar, 38(3), 237 - 45 Hydrolytic activities of human pancreatic phospholipase A2 and endotoxin-stimulated endogenous phospholipase A2 toward membrane phospholipids of erythrocytes; Nishijima J et al.; The problem of whether human pancreatic phospholipase A2 (PLA2) can really hydrolyze membrane phospholipids in vitro was studied to understand pathophysiology of acute pancreatitis . Total amount of lysophospholipids generated in erythrocytes by exogenously added human pancreatic PLA2 (2 micrograms/ml) was only 12% of the amount of sphingomyelin, which was not decomposed by the enzyme . About fivefold the amount of lysophospholipids was generated in ghost membranes during one-sixth of the incubation time compared to that in intact erythrocyte membranes . Escherichia coli lipopolysaccharide (LPS) (10 micrograms/ml) was able to stimulate membrane-associated PLA2 of erythrocytes, the amount of lysophospholipids generated being 12.5% of that of sphingomyelin without adding the exogenous PLA2 . The stimulation of membrane-associated PLA2 in erythrocytes was inhibited by pretreatment of lipopolysaccharide with polymyxin-B sulfate . When intact erythrocytes were incubated with human pancreatic PLA2 and LPS, the amount of generated lysophospholipids was 24% of that of sphingomyelin . These results suggested that the exogenously added human pancreatic PLA2 cannot degrade phospholipids of intact erythrocytes so extensively under physiological conditions, and, in acute pancreatitis, unknown factors may be involved in the hydrolysis of phospholipids . LPS, which activates membrane-associated PLA2, may be one of the factors, and thus membrane phospholipids are hydrolyzed in the disease. J Trauma, 1985 Mar, 25(3), 234 - 7 Sublethal hemorrhage impairs the acute peritoneal inflammatory response in the rat; Fink MP et al.; Hemorrhagic shock increases the risk of septic complications in injured patients . In this study, we investigated the effect of sublethal hemorrhage on the acute peritoneal inflammatory response and the clearance of bacteria from the peritoneal cavity of the rat . Sprague-Dawley rats were subjected to sublethal hemorrhage, resuscitated, and then inoculated intraperitoneally with a suspension of viable Escherichia coli in saline . Sham-hemorrhaged rats served as controls . Sublethal hemorrhage decreased survival and impaired the influx into the peritoneum of polymorphonuclear leukocytes and macrophage colony-forming cells . There was no difference between groups in the clearance of viable bacteria from the peritoneum; clearance of blood-borne bacteria was decreased in the hemorrhaged animals . We conclude that sublethal hemorrhage in the rat inhibits the acute peritoneal inflammatory response, but has little or no effect on the early removal of bacteria from the peritoneal cavity. Hepatology, 1985 Mar-Apr, 5(2), 192 - 7 T lymphocyte sensitization to HBcAg and T cell-mediated unresponsiveness to HBsAg in hepatitis B virus-related chronic liver disease; Vento S et al.; Using a newly developed indirect T lymphocyte migration inhibition test, cell-mediated immunity to HBsAg and HBcAg was directly and simultaneously examined in a total of 21 patients with HBsAg-positive chronic liver disease (CLD), and in seven subjects whose sera contained anti-HBs (2 previous acute hepatitis B; 4 hepatitis B vaccine recipients and 1 chronic active hepatitis) . T cell sensitization to HBcAg was invariably detected in the HBsAg-positive CLD patients tested (12/12), whereas T cell sensitization to HBsAg was not present in any of the patients (0/21) . In contrast, T cell sensitization to HBsAg was present in all anti-HBs-positive subjects . These results support the hypothesis that the cellular immune response to HBcAg, rather than to HBsAg, is implicated in the pathogenesis of HBsAg-positive CLD . Moreover, the observation that the addition of T cells from patients with HBsAg-positive CLD to T cells from anti-HBs positive subjects in a ratio of 1 to 9 reversed their sensitization to HBsAg, suggests that a hyperactivity of HBsAg-specific suppressor T cell population may be responsible for persistent HBs antigenemia. Am J Physiol, 1985 Mar, 248(3 Pt 2), R331 - 8 Alteration of adipocyte calcium homeostasis by Escherichia coli endotoxin; Nelson KM et al.; The present study evaluated calcium homeostasis in rat adipocytes after either in vivo or in vitro exposure to Escherichia coli endotoxin . Fat cells from endotoxin-treated rats showed an enhanced uptake of 45Ca . In an attempt to differentiate between 45Ca binding to the cell surface and intracellular 45Ca accumulation, adipocytes were exposed to 5 mM LaCl3 . The amount of 45Ca remaining associated with lanthanum-treated adipocytes was taken to be located intracellularly and was increased in adipocytes from endotoxin-treated rats . The amount of 45Ca displaced by lanthanum was also increased in adipocytes from endotoxin-treated rats . This suggested that the endotoxin-induced increase of 45Ca accumulation included both cell surface and intracellular binding sites . Compartmental analysis of the exchange kinetics of cell-associated 45Ca with 40Ca in the medium indicated a 77% increase in the size of the cell surface compartment of adipocytes from endotoxin-treated rats compared with controls . In addition, endotoxin treatment altered the flux of calcium from the cells to the medium . In vitro exposure of freshly prepared adipocytes to 250 or 750 micrograms endotoxin/ml did not produce a perturbation of adipocyte calcium homeostasis . The results indicate that endotoxin induces alterations in the ability of adipocytes to regulate calcium translocations, suggesting that some metabolic and hormonal aspects of endotoxins' actions may be mediated through perturbation of cellular calcium homeostasis. Proc Natl Acad Sci U S A, 1985 Mar, 82(5), 1480 - 4 Mutations in direct repeat sequences and in a conserved sequence adjacent to the repeats result in a defective replication origin in plasmid R6K; McEachern MJ et al.; Plasmid pMM3 is a pBR322 derivative carrying the gamma origin of replication of the naturally occurring plasmid R6K . We have produced a gamma-origin mutant bank of this plasmid using the single-strand-specific mutagen sodium bisulfite . Members of this bank contain single or multiple mutations in the seven direct repeats and the flanking sequences in the gamma origin . Three mutants with defective gamma origins have been isolated from this mutant bank . Two of these direct repeat mutants, gamma 117 and gamma 120, are unable to replicate and also have lost the ability to bind the R6K initiation protein pi in vitro at one of the seven 22-base-pair direct repeats within their respective origins . Precise deletion of the damaged repeat of either of these mutants restores origin function, suggesting that the primary defect of these mutants involves a disruption of the normal spacing of pi binding and flanking sequences within the gamma origin . The third mutant, gamma 111, binds pi normally but replicates at a greatly reduced copy number due to a mutation near the seventh repeat . This mutation falls within a short sequence that appears to be conserved among a number of other plasmids that contain direct repeats within their origins of replication. Proc Natl Acad Sci U S A, 1985 Mar, 82(5), 1367 - 71 Detection and characterization of a mouse alpha-spectrin cDNA clone by its expression in Escherichia coli; Cioe L et al.; A cloned segment of mouse alpha-spectrin mRNA has been identified by immunological techniques . Double-stranded cDNA derived from spleens of anemic mice was introduced into a bacterial expression vector, pUC, and transformed Escherichia coli colonies were screened by using an antiserum to erythrocyte membrane ghost proteins . Of 17 positive colonies, 2 bound antibody to mouse spectrin, and these 2 colonies contained 750-base-pair inserts that cross-hybridized . Transfer of the 750-base-pair insert to an expression vector containing the PL promoter of phage lambda produced larger amounts of peptides that were bound by antibody to mouse spectrin . The spectrin-like peptides made in E . coli elicited antibody that reacted only with the alpha-spectrin subunit of erythrocyte membranes . This clone will be useful for the study of the structure and expression of the spectrin gene, particularly in understanding the role of spectrin in human inherited hemolytic anemias. Proc Natl Acad Sci U S A, 1985 Mar, 82(5), 1326 - 30 Chimeric chemosensory transducers of Escherichia coli; Krikos A et al.; The tar and tsr genes of Escherichia coli encode homologous transducer proteins that mediate distinct chemotactic responses . We report here the construction of two tasr chimeric genes in which the 5' coding region of the tar gene is fused to the 3' coding region of the tsr gene at either of two conserved restriction sites . Both chimeric genes code for chemotactically functional proteins . Results of analyses of behavior and methylation in cells carrying the chimeric genes support existing models for the disposition of transducer domains across the cell membrane and reveal that the receptors for internal pH map in a specific region of the COOH-terminal (cytoplasmic) domain. Obstet Gynecol, 1985 Mar, 65(3 Suppl), 84S - 87S Malakoplakia of the female genital tract; Chen KT et al.; A rare case of malakoplakia of the uterine cervix and the pelvis occurring in an elderly woman who also had xanthogranulomatous pyelonephritis is described and compared with the 15 reported cases of malakoplakia of the female genital tract. J Med Chem, 1985 Mar, 28(3), 333 - 46 Use of physicochemical parameters in distance geometry and related three-dimensional quantitative structure-activity relationships: a demonstration using Escherichia coli dihydrofolate reductase inhibitors; Ghose AK et al.; In earlier distance geometry related three-dimensional quantitative structure-activity relationships (Ghose, A . K.; Crippen, G . M . J . Med . Chem . 1984, 27, 901) the interactions of the ligand atom or group with the receptor site were evaluated empirically by using mathematical optimization techniques, without considering their physicochemical properties . In the present work we show how to use various physicochemical parameters in our three-dimensional receptor mapping . We have developed a model for E . coli DHFR using the inhibition data of 25 pyrimidines and 14 triazines . It gave a correlation coefficient of 0.893 and standard deviation of 0.530 . It successfully predicted the binding data of five pyrimidines and five triazines. J Infect Dis, 1985 Mar, 151(3), 471 - 5 A newly recognized cause of travelers' diarrhea: enteroadherent Escherichia coli; Mathewson JJ et al.; Adherence to HEp-2 tissue culture cells has been proposed as a virulence characteristic of enteropathogenic Escherichia coli (EPEC) . A preliminary study revealed that E . coli that adhered to HEp-2 cells, but did not produce conventional enterotoxins and did not belong to recognized EPEC serogroups, could be isolated from adults from the United States who acquired diarrhea in Mexico . The purpose of this study was to determine the prevalence of these enteroadherent E . coli (EAEC) in 188 travelers with diarrhea and in 92 well travelers . EAEC were found in 14.9% of patients with diarrhea and in 7.6% of well individuals . Compared with well travelers, patients with diarrhea in whom no recognized enteropathogen could be identified had a 30.4% prevalence of EAEC (P less than .0003) . These results further support our finding that EAEC are associated with diarrhea in travelers to Mexico and may help to explain the effect of antibiotics in the prevention and therapy for travelers' diarrhea in patients with no recognized bacterial enteropathogens. J Comput Assist Tomogr, 1985 Mar-Apr, 9(2), 280 - 7 Computed tomography of nontuberculous spinal infection; Whelan MA et al.; The CT findings in 16 patients with nontuberculous spinal infections were reviewed . The specificity of certain CT features as well as the usefulness of intravenous contrast medium administration are discussed . The associated clinical presentations and predisposing factors are outlined . Emphasis is placed on a combined clinical, radiographic approach in facilitating an early diagnosis. J Bacteriol, 1985 Mar, 161(3), 973 - 80 Alkaline phosphatase and OmpA protein can be translocated posttranslationally into membrane vesicles of Escherichia coli; Chen L et al.; We previously described a system for translocating the periplasmic enzyme alkaline phosphatase and the outer membrane protein OmpA into inverted membrane vesicles of Escherichia coli . We have now optimized and substantially improved the translocation system by including polyamines and by reducing the amount of membrane used . Under these conditions, efficient translocation was seen even posttranslationally, i.e., when vesicles were not added until after protein synthesis was stopped . This was the case not only with the OmpA protein, which is synthesized by free polysomes and hence is presumably exported posttranslationally in the cell, but also with alkaline phosphatase, which is synthesized only by membrane-bound polysomes and has been shown to be secreted cotranslationally in the cells . Prolonged incubation rendered the precursors inactive for subsequent translocation . Posttranslational translocation was impaired, like cotranslational translocation, by inhibitors of the proton motive force and by treatment of the vesicles with protease . Since it appears that E . coli can translocate the same proteins either cotranslationally or posttranslationally, the cotranslational mode may perhaps be more efficient, but not obligatory, for the secretion of bacterial proteins. J Bacteriol, 1985 Mar, 161(3), 949 - 54 Accumulation of prolipoprotein in Escherichia coli mutants defective in protein secretion; Hayashi S et al.; The export of lipoprotein has been found to be affected in both secA and secY mutants of Escherichia coli which are defective in the secretion of a number of outer membrane and periplasmic proteins . The kinetics of accumulation of prolipoprotein upon a temperature shift to 42 degrees C is indistinguishable from that of pre-OmpA protein accumulation in the secA mutant . In both secA and secY mutants, the accumulated prolipoprotein is unmodified with glyceride and localized in the cytoplasmic membrane . We conclude from these results that the early steps in protein export are common to prolipoprotein and non-lipoprotein precursors . The pathways for the export of these two groups of precursor proteins diverge with regard to the modification and processing reactions which are late events in the export process. J Bacteriol, 1985 Mar, 161(3), 939 - 43 Mutations in the rpoH (htpR) gene of Escherichia coli K-12 phenotypically suppress a temperature-sensitive mutant defective in the sigma 70 subunit of RNA polymerase; Grossman AD et al.; Escherichia coli K-12 strain 285c contains a mutation in rpoD, the gene encoding the sigma subunit of RNA polymerase . The 70-kilodalton sigma polypeptide encoded by this allele is unstable, and this instability leads to temperature-sensitive growth . We describe the isolation and characterization of four temperature-resistant pseudorevertants of 285c that can grow at high temperature . Each of these revertants increased the stability of the sigma 70 mutant protein . The map position of the suppressor mutations was close to that of the rpoH (htpR) gene . A multicopy plasmid containing the intact rpoH gene restored the temperature-sensitive phenotype . Marker rescue experiments established the positions of three of the alleles within the rpoH gene . One mutation has been sequenced and causes a leucine-to-tryptophan change 7 amino acids from the carboxyl terminus of the rpoH gene product. J Bacteriol, 1985 Mar, 161(3), 933 - 8 Single-strand breakage of DNA in UV-irradiated uvrA, uvrB, and uvrC mutants of Escherichia coli; Tang MS et al.; We transduced the uvrA6, uvrB5, uvrC34, and uvrC56 markers from the original mutagenized strains into an HF4714 background . Although in the original mutagenized strains uvrA6 cells are more UV sensitive than uvrB5 and uvrC34 cells, in the new background no significant difference in UV sensitivity is observed among uvrA6, uvrB5, and uvrC34 cells . No DNA single-strand breaks are detected in UV-irradiated uvrA6 or uvrB5 cells, whereas in contrast a significant number of single-strand breaks are detected in both UV-irradiated uvrC34 and uvrC56 cells . The number of single-strand breaks in these cells reaches a plateau at 20-J/m2 irradiation . Since these single-strand breaks can be detected by both alkaline sucrose and neutral formamide-sucrose gradient sedimentation, we concluded that the single-strand breaks observed in UV-irradiated uvrC cells are due to phosphodiester bond interruptions in DNA and are not due to apurinic/apyrimidinic sites. J Bacteriol, 1985 Mar, 161(3), 904 - 8 Nucleotide sequence of the gene for the vitamin B12 receptor protein in the outer membrane of Escherichia coli; Heller K et al.; The nucleotide sequence of a 2220-base-pair fragment containing the btuB gene of Escherichia coli was determined . There was a single open reading frame which was translated into a 614-amino-acid polypeptide, the first 20 amino acids of which comprised a typical leader sequence . The putative mature or processed form had a molecular weight (66,400) and a composition in close agreement with that determined for the purified receptor . The distribution of amino acids in the receptor protein was similar to that of other outer membrane proteins, showing a fairly even distribution of charged residues and the absence of extensive hydrophobic stretches . The btuB451 mutation appears to alter the receptor to eliminate its ability to function in vitamin B12 uptake without affecting its ligand binding properties . The sequence of the DNA from this mutant was determined and revealed a leucine-to-proline (C-to-T transition) change in the eighth amino acid of the mature form. J Bacteriol, 1985 Mar, 161(3), 1209 - 14 New cysE-pyrE-linked rfa mutation in Escherichia coli K-12 that results in a heptoseless lipopolysaccharide; Coleman WG Jr et al.; A new novobiocin-supersensitive mutant of Escherichia coli K-12 has been characterized biochemically and genetically . Lipopolysaccharide prepared from this mutant strain is truncated and contains 2-keto-3-deoxyoctulosonic acid as its only core sugar . This new core-defective mutation, designated rfa-2, results in increased sensitivity to several hydrophobic and some hydrophilic agents . Genetic analysis of the rfa mutant indicated that the rfa-2 locus is located at 81 min on the chromosome . The order of the genes in this region based on transduction analysis is xyl cysE rfa-2 rfaD70 pyrE . P1 transduction analyses indicate that the rfa-2 marker is nonallelic with the recently described cysE-pyrE-linked rfaD70 locus . Plasmids carrying the wild-type rfaD70+ allele failed to abolish the rfa-2 phenotypes . Further, the rfaD gene product, ADP-L-glycero-D-mannoheptose-6-epimerase, was detected in crude extracts of a rfa-2 mutant strain, CL609, and was absent in the rfaD70 mutant . The wild-type rfa-2 allele codes either for a specific heptose biosynthetic enzyme (different from the rfaD gene product) or an enzymatic activity required for the addition of heptose to the lipid A-2-keto-3-deoxyoctulosonic acid acceptor. J Bacteriol, 1985 Mar, 161(3), 1059 - 68 Overproduction and nucleotide sequence of the respiratory D-lactate dehydrogenase of Escherichia coli; Rule GS et al.; Recombinant DNA plasmids containing the gene for the membrane-bound D-lactate dehydrogenase (D-LDH) of Escherichia coli linked to the promoter PL from lambda were constructed . After induction, the levels of D-LDH were elevated 300-fold over that of the wild type and amounted to 35% of the total cellular protein . The nucleotide sequence of the D-LDH gene was determined and shown to agree with the amino acid composition and the amino-terminal sequence of the purified enzyme . Removal of the amino-terminal formyl-Met from D-LDH was not inhibited in cells which contained these high levels of D-LDH. J Bacteriol, 1985 Mar, 161(3), 1054 - 8 Pi exchange mediated by the GlpT-dependent sn-glycerol-3-phosphate transport system in Escherichia coli; Elvin CM et al.; The GlpT system for sn-glycerol-3-phosphate transport in Escherichia coli is shown to catalyze a rapid efflux of Pi from the internal phosphate pools in response to externally added Pi or glycerol-3-phosphate . A glpR mutation, which results in constitutive expression of the GlpT system, is responsible for this rapid Pi efflux and the arsenate sensitivity of several laboratory strains, including the popular strain C600 . Glucose and other phosphotransferase system sugars inhibit Pi efflux by repressing glpT expression. J Bacteriol, 1985 Mar, 161(3), 1049 - 53 Mutant of Escherichia coli deficient in osmoregulation of periplasmic oligosaccharide synthesis; Clark DP; A mutant of Escherichia coli (mdoR) has been isolated which is defective in synthesis of the membrane-derived oligosaccharides (MDO) normally found in the periplasmic space . In media of high osmotic pressure this defect is suppressed and MDO levels approaching those of the wild type are produced . The mdoR mutant also fails to accumulate glycogen; however, genetic analysis showed that mdoR was not cotransducible with the known glg (glycogen) locus . A further relationship between MDO and glycogen metabolism was suggested by two observations that (i) certain glg mutants affect MDO accumulation and (ii) elevated osmotic pressure inhibits glycogen accumulation, in both wild-type and mdoR cells. J Bacteriol, 1985 Mar, 161(3), 1023 - 8 Escherichia coli mutant with altered respiratory control of the frd operon; Iuchi S et al.; In wild-type Escherichia coli, fumarate reductase encoded by the frd operon is inducible by its substrate in the absence of molecular oxygen and nitrate . Synthesis of this enzyme under permissive conditions requires the fnr+ gene product, which is believed to be a pleiotropic regulatory protein that activates transcription . A spontaneous mutant was isolated in which the expression of the frd operon no longer depended on the presence of fumarate or the fnr+ gene product . Aerobic repression of the operon was abolished, but nitrate repression remained intact . Transductional analysis showed that the mutation was closely linked to the frd locus . The mutant phenotype strongly suggests that repression by molecular oxygen and nitrate is mediated by different mechanisms. Infect Immun, 1985 Mar, 47(3), 834 - 6 Revised amino acid sequence for a heat-stable enterotoxin produced by an Escherichia coli strain (18D) that is pathogenic for humans; Thompson MR et al.; The amino acid sequence of heat-stable enterotoxin produced by enterotoxigenic Escherichia coli 18D has been revised . Amino acids originally assigned to positions 11 and 18, i.e., Tyr and Asn, respectively, were found to occupy positions 18 and 11, respectively . Thus all heat-stable enterotoxins composed of 18 amino acids sequenced to date from human, porcine, and bovine isolates of E . coli have identical primary structures, i.e., Asn-Thr-Phe-Tyr-Cys-Cys-Glu-Leu-Cys-Cys-Asn-Pro-Ala-Cys-Ala-Gly-Cys-Tyr . Furthermore, all 18- and 19-amino-acid heat-stable enterotoxins from E . coli share an almost identical core sequence, i.e., 14 of the 15 carboxy-terminal amino acid residues are identical. Infect Immun, 1985 Mar, 47(3), 665 - 9 Activation of mouse peritoneal adherent cells with N-acyl muramyl dipeptide derivatives; Nishimura K et al.; The effect of N-acyl derivatives of muramyl dipeptide (N-acetyl muramyl-L-alanyl-D-isoglutamine) on the activation of peritoneal adherent cells (PAC) in vivo and on the stimulation of nonspecific host resistance against Escherichia coli infection was examined in comparison with the effect of 6-O-stearoyl muramyl dipeptide . N-acyl muramyl dipeptide derivatives increased the release of hydrogen peroxide (H2O2) by PAC from mice treated 1 day before upon stimulation with phorbol myristate acetate, and their activities did not depend on the chain length or kinds of fatty acids introduced . The results obtained using N-stearoyl muramyl dipeptide analogs indicated that the acyl moiety combined to muramic acid played a more important role in the ability of PAC to release H2O2 than did the peptide moiety . PAC from mice treated with N-stearoyl muramyl dipeptide, N-(3-hydroxy-2-docosylhexacosanoyl) muramyl dipeptide, and 6-O-stearoyl muramyl dipeptide 1 day before, including 20 to 42% polymorphonuclear leukocytes, released large amount of H2O2, and most of the H2O2 released was due to the attribution of polymorphonuclear leukocytes . The cytostatic activity of PAC from mice treated with these three compounds reached a maximum on day 3 after injection, and the cytolytic activity of PAC was induced by N-stearoyl muramyl dipeptide on day 3 and by 6-O-stearoyl muramyl dipeptide on day 1 after injection . In contrast to the above results, N-acyl muramyl dipeptide derivatives did not stimulate nonspecific host resistance against E . coli infection in mice when compared to 6-O-stearoyl muramyl dipeptide. Eur J Biochem, 1985 Mar 1, 147(2), 381 - 6 Mechanism of action of gentamicin components . Characteristics of their binding to Escherichia coli ribosomes; Tangy F et al.; The binding of gentamicin (Gm) to Escherichia coli ribosomes and ribosomal subunits has been studied . By means of equilibrium dialysis and of statistical interpretation of the data it was found that {3H}gentamicin C2 and 6'-N-{3H}methylgentamicin C1a interact with three classes of sites on tight-coupled 70-S species: a first class concerning the tight and non-cooperative interaction with one drug molecule (Kd = 0.6 microM), a second class in which about five Gm molecules bind cooperatively (mean Kd = 10 microM), and a third class of very high capacity in which up to 70 drug molecules may interact . The extreme cooperativity of the third class of sites induces such an increase in the affinity for Gm that it may allow the shift of molecules already bound from high-affinity sites towards lower-affinity sites . The alteration of a ribosomal protein, L6, in a gentamicin-resistant mutant of E . coli abolished the multiclass and the cooperative aspects of ribosomes--gentamicin interaction . The large ribosomal subunits from E . coli MRE 600 strain interact cooperatively with Gm, whereas 50-S particles from the resistant mutant bind the drug in a diffuse way with high capacity and low affinity . The small subunits from both strains behave identically towards Gm . A good correlation is observed in comparing the gentamicin concentrations capable of saturating the different ribosomal classes of sites with concentrations inducing its multiphasic effects on protein synthesis. Eur J Biochem, 1985 Mar 1, 147(2), 325 - 9 Location of the adenylylation site in T4 RNA ligase; Thogersen HC et al.; The purification of the enzyme T4 RNA ligase is described from an Escherichia coli strain, KR54, in which the RNA ligase gene (g63) has been inserted into the plasmid pDR540 for inducible expression of g63 from the tac promoter . Adenylylation of the purified enzyme with {14C}rATP followed by digestion with chymotrypsin yielded an adenylylated peptide, the identity of which was determined by fast-atom-bombardment mass spectrometric analysis . The results show that the AMP residue is bound covalently to the lysine at position 99 of the RNA ligase protein sequence. Proc Natl Acad Sci U S A, 1985 Mar, 82(5), 1536 - 9 Isolation of a cDNA clone encoding pancreatic polypeptide; Takeuchi T et al.; We have isolated a cDNA clone encoding pancreatic polypeptide from a cDNA library constructed with RNA from an endocrine neoplasm of the human pancreas . The cDNA was inserted into plasmid pBR322 and the plasmid was cloned in Escherichia coli . Oligonucleotides (sequence in text) specific for the amino acid sequence (sequence in text) of pancreatic polypeptide were used as hybridization probes . The pancreatic polypeptide cDNA isolated was 465 base pairs long and encoded a peptide of 95 amino acids in the coding region . The 36-amino acid sequence of pancreatic polypeptide was flanked by a 29-amino acid putative leader sequence at the amino terminus and a connecting tripeptide (Gly-Lys-Arg) followed by a 27-amino acid peptide at the carboxyl terminus . The first 20 of the amino acids in the carboxyl-terminal heptacosapeptide were identical to the structure of human pancreatic icosapeptide with the single exception of an isoleucine substitution for valine in the 18th position . This alteration results from an A----G substitution in the nucleotide sequence of the cDNA and may represent a genetic variation or a point mutation in the pancreatic polypeptide cDNA. Proc Natl Acad Sci U S A, 1985 Mar, 82(5), 1401 - 5 A minimal ribosomal RNA: sequence and secondary structure of the 9S kinetoplast ribosomal RNA from Leishmania tarentolae; de la Cruz VF et al.; The portion of the Leishmania tarentolae kinetoplast maxicircle DNA encoding the 9S RNA gene was sequenced, and the 5' and 3' ends of the transcript were determined . A secondary structure for the 9S RNA was determined based on the Escherichia coli 16S model . The 610-nucleotide 9S RNA exhibits a minimal secondary structure in which all four domains of the E . coli 16S structure are preserved . Within domains, however, some stems and loops have been greatly reduced or eliminated entirely . It is presumed that these reduced domains represent the minimal essential small ribosomal RNA secondary structures necessary for a functional ribosome . Alignment of the L . tarentolae 9S rRNA sequence with the published Trypanosoma brucei 9S rRNA sequence shows a nucleotide similarity of 84% and a transversion/transition ratio of 1.66. J Med Chem, 1985 Mar, 28(3), 323 - 7 Artificial siderophores . 2 . Syntheses of trihydroxamate analogues of rhodotorulic acid and their biological iron transport capabilities in Escherichia coli; Lee BH et al.; Tris{(acetylhydroxyamino)alkyl} isocyanurates 2a-c were synthesized from alpha, omega-dibromoalkanes 5 in four steps . The alkylation of the bromides 5a-c with O-benzyl-N-{(trichloroethoxy)carbonyl}hydroxylamine in the presence of DBU gave N-alkylation products 7a-c . The (trichloroethoxy)cabronyl protecting group of 7a-c was easily removed by Zn dust in acetic acid . When the reaction was performed with acetic anhydride, the desired N-acetylated materials 10a-c were obtained . The alkylation of cyanuric acid with 12 in the presence of base provided the N-alkylated materials 13, which were hydrogenated to provide 2a-c . In order to determine the affect of structural modifications on biological activity, various chain lengths of the side arms were utilized and the retroanalogue 3 was prepared . Most of the compounds examined acted as ferrichrome in supporting the iron nutrition of Escherichia coli . However, tris{(acetylhydroxyamino)butyl} isocyanurate 2b, and to some extent its pentyl analogue, 2c, displayed the unique and remarkable property of supporting growth of fhuB mutants, the latter unresponsive to the other analogues and to all natural siderophores tested. J Bacteriol, 1985 Mar, 161(3), 1201 - 8 Essential and nonessential sequences in malPp, a positively controlled promoter in Escherichia coli; Raibaud O et al.; A plasmid bearing the malPp promoter was digested with Bal31 to obtain a set of deletions with closely spaced endpoints in the upstream region of this promoter . Some of these deletions were sequenced, and their effect on malPQ expression was determined after having transferred them onto the chromosome . We found that a site which binds the cyclic AMP receptor protein in vitro and which is centered at position -93 with respect to the site of transcription initiation could be deleted without affecting malPQ expression . In contrast, the activity of the malPp promoter decreased abruptly when the deletions reached position -72 . The downstream region of the promoter was analyzed by using a technique of "sequence replacement" which involved the selection of Mal+ pseudorevertants from strains which carried small deletions in the -25 region . The pseudorevertants, which expressed the malPQ operon in a manner indistinguishable from wild type, had grossly different sequences downstream from position -38, except for a few positions, some of which must be important for promoter function . By combining all presently available information, it is suggested that the malPp promoter contains three binding sites for its activator, the product of gene malT . These sites are defined by three quasi-identical hexanucleotides present in one orientation around position -37 and twice in the other orientation around positions -60 and -73. J Bacteriol, 1985 Mar, 161(3), 1080 - 5 Efficiency of induction of prophage lambda mutants as a function of recA alleles; Dutreix M et al.; Mutants of the cI gene of prophage lambda have been defined phenotypically in a recA+ host as noninducible (Ind-), inducible (Ind+), or induction sensitive (Inds) . We showed that a phage lambda cI+ carrying operator mutations v2 and v3 displays an Inds phenotype, as does lambda cI inds-1 . We characterized a fourth induction phenotype called induction resistant (Indr) . Using these four prophage types, we tested the influence of bacterial recA mutations on prophage induction . Indr prophages were fully induced in recA441 bacteria whose RecA441 protein is activated constitutively . Indr prophages were not induced in a mutant overproducing RecA+ protein, confirming that RecA+ protein must be activated to promote prophage induction . Inds prophages were induced in recA142 and recA453-441 lysogens, previously described as deficient in prophage induction. Mol Gen Mikrobiol Virusol, 1985 Mar, (3), 19 - 22 {Restriction mapping of genetic transfer factor pAP39}; Pekhov AP et al.; A model of calculation of molecular weights of fragments EcoRI, Hind III and PvuI is formulated . A restriction site map of factor pAP39 is constructed automatically . Sites to EcoRI and PvuI are distributed in the segment with molecular weight 9.1 MD. Exp Eye Res, 1985 Mar, 40(3), 411 - 9 Ocular responses to superoxide generated by intraocular injection of xanthine oxidase; Mittag TW et al.; Xanthine oxidase (XaO) was injected into the anterior chamber of rabbit eyes by a closed circuit perfusion system . Doses of 1.5 milliunits (mU) or greater produced a maximal leucocyte accumulation after 4 hr, with an initial elevation of ocular pressure in the first 15 min . Similar experiments on rats with intravitreal injections of 0.1-1.5 mU of XaO resulted in a significant accumulation of leucocytes after 5 hr which, at the highest dose of XaO, was partly due to traces of bacterial endotoxin in the XaO . However, in endotoxin-desensitized rats the response to 1.5 milliunits XaO was seven-fold greater than the response to endotoxin alone . Simultaneous administration of xanthine (Xa) substrate with XaO was not required to elicit cell infiltration into the anterior chamber . Dialyzed enzyme was also effective but boiling abolished the response . Addition of XaO to rabbit aqueous humor in vitro decreased the ascorbate content, consistent with the generation of superoxide from an endogenous substrate . The results suggest that enzymatically active XaO, which can cause intraocular generation of superoxide from an XaO substrate present in aqueous humor, initiates the chemotactic response . A chemotactic agent may be generated from superoxide reacting with endogenous precursors in aqueous humor or by selective activation of the lipoxygenase pathway of arachidonic acid metabolism in adjacent tissues. Antibiot Med Biotekhnol, 1985 Mar, 30(3), 166 - 70 {Cis-active function of plasmid R 57 resolving co-integrates formed during transposition of ISI-elements}; Danilevich VN; The mechanism of pBR322 plasmid mobilization in the cells of Escherichia coli K-12 recA by conjugative factor R57 was studied . It was shown that mobilization of pB322 is achieved by formation of unstable IS1-mediated cointegrates with R57 . In the rec+ E . coli strains cointegrates resolved with formation of pBR322:IS1 plasmids . In the recA bacteria the cointegrates dissociated to pBR:IS1, as well as to other insertion derivatives of pBR322 . Some of the latter contain Tn9-like sequence, i.e . a transposon flanked by direct repeats of IS1 . The subsequent transposition of IS1 from pBR- . IS1 to pBR3.1 plasmid (Aps deletion derivative of RP1) leads to formation of stable cointegrates incapable of dissociating even in the presence of coresident plasmid R57 . It is suggested that R57 encodes the cys-acting function providing recA-independent recombination between direct repeats of IS1. Plasmid, 1985 Mar, 13(2), 88 - 98 Identification of an Rts1 DNA fragment conferring temperature-dependent instability to vector plasmids; Okawa N et al.; The multiphenotypic drug resistance factor Rts1 expresses a temperature-dependent instability characteristic . This plasmid was digested with the restriction enzyme BamHI . A DNA fragment with a molecular weight of 5.6 MDa (the H fragment) was inserted into plasmid pBR322 (pFK896) or into pSC105 (pYH156) at the BamHI site . These plasmids were unstable at 42 degrees C but stable at 32 degrees C . A restriction-enzyme map of the H fragment was constructed and the instability phenotype (Tdi) was localized to a DNA fragment with 0.5 MDa molecular weight . The temperature-dependent loss of the unstable plasmid pFK896 is abrupt and no gradual plasmid loss of this multicopy recombinant plasmid is observed . The possibility that the Tdi phenotype is due to overgrowth of R- cells was eliminated. Plasmid, 1985 Mar, 13(2), 145 - 8 Location of rep and inc sequences in the F secondary replicon; Gardner R et al.; Miniplasmids derived by deletion of DNA from the F plasmid secondary replicon have been tested for the ability to replicate and to express incompatibility with the IncFI plasmid, ColV3-K30 . The results demonstrate that the minimal rep region of the secondary replicon lies within a 1.9-kb sequence (33.7F-35.6F kb), and that an inc region, presumably involved in replication control, is present in a 0.45-kb portion of the rep region (33.7F-34.15F kb) . In addition, the secondary replicon was found not to require DNA polymerase I activity. Am J Vet Res, 1985 Mar, 46(3), 587 - 90 Dose-response evaluation of a genetically engineered foot-and-mouth disease virus polypeptide immunogen in cattle; McKercher PD et al.; Four groups of 9 cattle each were vaccinated with 10, 50, 250, or 1,250 micrograms of foot-and-mouth disease (FMD) virus A12 VP1 fusion protein that was produced in Escherichia coli and emulsified in an oil adjuvant . The groups given the 10 and 50 micrograms of antigen were revaccinated at 15 weeks and were challenge exposed at 30 weeks; 5 of 9 and 7 of 9 cattle, respectively, were protected from FMD virus infection . The remaining 2 groups, vaccinated with 250 or 1,250 micrograms of antigen, were revaccinated at 32 weeks and were challenge exposed at 45 weeks; 8 of 9 and 9 of 9 cattle, respectively, were protected . The results indicated that the biosynthetic polypeptide FMD vaccine was effective using vaccination intervals frequently followed with conventional whole-virus vaccines. Mol Cell Biol, 1985 Mar, 5(3), 529 - 37 Rapid assay for extrachromosomal homologous recombination in monkey cells; Rubnitz J et al.; Most of the recombination assays based on the regeneration of selectable marker genes after transient infection or stable integration of DNA into mammalian cells are time consuming . We have used plasmids containing two truncated but overlapping segments of the neomycin resistance gene to rapidly quantitate and characterize the time course of extrachromosomal homologous recombination of DNA transfected into monkey COS cells . By transiently infecting cells with these recombination substrates, extracting Hirt DNA after 1 to 4 days, and transforming recombination-deficient Escherichia coli, we have shown that recombination between direct repeats occurs at frequencies of 1 to 4% . We have also used Southern blot analysis to directly characterize the recombination of this DNA in COS cells and to demonstrate that double-strand breaks in the region of homology increase recombination frequencies 10- to 50-fold. Genetika, 1985 Mar, 21(3), 375 - 83 {Chromosomal inversion accompanied by an enhancement of uridine phosphorylase gene expression in Escherichia coli K-12}; Kulakauskas ST et al.; In thymine requiring auxotrophs of Escherichia coli the uridine phosphorylase enzyme (udp gene) can catalyze nonspecifically conversion of thymine to thymidine . By selection for effective utilization of exogenous thymine, it is possible to isolate forms with increased expression of the udp gene . Mutants with increased gene expression were isolated from the strain with transposon Tn10 within the metE gene closely linked to udp . Some mutants (designated udpPf) losing Tn10 but retaining the Met- phenotype are characterized by disturbance of recombination in the metE-udp region: they do not form Met+ transductants in P1 transduction with the wild-type donor strain . However, recovery of homology in the chromosomal metE-udp region takes place with low frequency in P1 transduction using the strain with Tn10 insertion in metE as a donor . Data obtained in transductional and conjugational experiments demonstrate that the udpPf1 mutant studied is an inversion extending about 3 min of the E . coli chromosome and including the region of chromosomal replication origin (oriC). Am J Hum Genet, 1985 Mar, 37(2), 311 - 25 A molecular basis for discrete size variation in human ribosomal DNA; Erickson JM et al.; The tandemly repeated human ribosomal RNA (rRNA) genes contain a region of size heterogeneity that is present in the nontranscribed spacer of every individual examined . This heterogeneity has been previously examined by Southern analysis of BamHI-digested human DNA . Using a ribosomal DNA (rDNA) probe specific for the 3' end of the 28S rRNA gene, at least four discrete sizes of BamHI fragments were seen in human populations . Molecular analysis of the cloned DNA from this region reveals tandem duplication of a segment of spacer rDNA located 388 base pairs (bp) 3' to the end of the 28S ribosomal RNA gene . Five hundred fifty bp of DNA, flanked on either side by a 150-bp repeated element, is either duplicated or deleted to produce a series of spacers that differ in size by 850 bp . These duplications/deletions appear to be the product of unequal homologous exchange, mediated by the small repeated element . Thus, human rDNA fragments cloned in lambda vectors and propagated in E . coli generate the same apparent size variation seen in genomic DNA . This study suggests that unequal homologous exchange is the molecular basis for the observed length heterogeneity in the spacer rDNA and may be a common mechanism for the generation of human genetic diversity. Vet Pathol, 1985 Mar, 22(2), 156 - 63 Dysentery caused by Escherichia coli (S102-9) in calves: natural and experimental disease; Hall GA et al.; A dysentery syndrome was recognized among the Institute's calves at 18 to 21 days of age . It was reproduced experimentally in gnotobiotic calves with an atypical Escherichia coli (S102-9) isolated from the affected calves . In both natural and experimental disease the calves passed copious bright red blood in the feces and developed diarrhea . Walls of the colon and rectum were thickened, and the mucosa was reddened and covered by an exudate that contained mucus and blood clots . Bacteria were seen closely adherent to the luminal surfaces of enterocytes, often in cup-shaped depressions or on cytoplasmic pedestals . Microvilli were distorted, disorientated or absent . There was exfoliation of infected enterocytes and a mild acute inflammation of the underlying lamina . In two of five calves with natural disease, the adherent bacteria did not stain by the immunoperoxidase method with antisera raised against E . coli (S102-9) . This indicated that there was possibly more than one bacterial cause of the syndrome . Lesions in experimentally infected calves were indistinguishable from those produced by some E . coli which are enteropathogenic for man, rabbits, and pigs. Surgery, 1985 Mar, 97(3), 300 - 7 Effect of hydroxyl radical scavenging on endotoxin-induced lung injury; Wong C et al.; The release of oxygen radicals, in particular the hydroxyl radical, from sequestered neutrophils produces acute lung injury after a number of insults . Our purpose was to determine whether hydroxyl radical, OH., is responsible for the lung injury from endotoxin characterized by (1) pulmonary leukostasis, (2) increased thromboxane production leading to pulmonary hypertension and hypoxia, and (3) increased protein permeability . This hypothesis was tested by infusion of a selective OH . scavenger, dimethyl thiourea (0.75 gm/kg), into unanesthetized sheep before endotoxin and comparison of the response to that seen with endotoxin alone . Pulmonary vascular integrity was measured by the use of lung lymph flow, QL, and lymph protein transport . Thromboxane A2 was measured as TxB2 and prostacyclin as 6-keto-PGF1 alpha . We found no difference in the degree of leukopenia and hypoxia after endotoxin or the levels of TxB2, 6-keto-PGF1 alpha, and pulmonary hypertension with dimethyl thiourea, compared with endotoxin alone . The permeability injury was also identical, with a twofold to threefold increase in protein-rich lymph seen in both groups . It appears that OH . does not play a major causative role in either phase of endotoxin lung injury. Proc Natl Acad Sci U S A, 1985 Mar, 82(5), 1381 - 5 Mapping the location of psoralen crosslinks on RNA by mung bean nuclease sensitivity of RNA.DNA hybrids; Hui CF et al.; An indirect high resolution method has been developed for finding the location of intrastrand crosslinks in RNA . An end-labeled DNA strand that overlaps the approximate crosslink position is hybridized to the RNA and then treated with mung bean nuclease . The resulting digest is analyzed on a sequencing-type gel . The method was tested with the major psoralen crosslink seen in the 16S rRNA of inactivated Escherichia coli 30S ribosomal subunits . This crosslink was previously mapped between residues 930 +/- 25 and a region close to the 3' end by electron microscopy . The new indirect method reveals that the crosslink occurs between residues 919 and 923 and residues 1530 and 1534 . When these results are examined in the light of existing consensus secondary structure models for the 16S rRNA, it appears that the Shine-Dalgarno sequence is located close to the peptidyl tRNA binding site. J Bacteriol, 1985 Mar, 161(3), 928 - 32 recA-independent recombination between repeated IS50 elements is not caused by an IS50-encoded function; Phadnis SH et al.; Certain pBR322-related plasmids containing direct repeats of the insertion element IS50 appear to be unstable in recA Escherichia coli because smaller recombinant derivatives accumulate rapidly in plasmid DNA populations . We show here that (i) this instability is plasmid specific, but not IS50 specific; (ii) it is due to a detrimental effect exerted by these plasmids on bacterial growth; and (iii) the growth impairment is alleviated in cells harboring the smaller recombinant plasmids . Although a recent report had concluded that accumulation of recombinants reflected an IS50-specific recombination function, when correction is made for the relative growth rates of cells containing the parental and recombinant plasmids the evidence for such a recombination function disappears. J Bacteriol, 1985 Mar, 161(3), 896 - 903 Cloning and expression of the gene for the vitamin B12 receptor protein in the outer membrane of Escherichia coli; Heller K et al.; The transport of cyanocobalamin (vitamin B12) in cells of Escherichia coli is dependent on a receptor protein (BtuB protein) located in the outer membrane . A 9.1-kilobase pair BamHI fragment carrying the btuB gene was cloned from a specialized transducing phage into multicopy plasmids . Insertions of transposon Tn1000 which prevented production of the receptor localized btuB to a 2-kilobase pair region . Further subcloning allowed isolation of this region as a 2.3-kilobase pair Sau3A fragment . The BtuB+ plasmids were shown by maxicell analysis to encode a polypeptide with a molecular weight of 66,000 in the outer membrane . This polypeptide was missing in cells with Tn1000 insertions in btuB and was reduced in amount upon growth of plasmid-bearing cells in repressing concentrations of vitamin B12 . Several Tn1000 insertions outside the 5' end of the coding region exhibited reduced production of receptor . A deletion at the 3' end of btuB resulted in formation of an altered receptor . Amplified production of this polypeptide was associated with increased levels of binding of the receptor's ligands (vitamin B12 and phage BF23), increased rates of vitamin B12 uptake, and altered susceptibility to the group E colicins . Deficiency in various major outer membrane proteins did not affect production of the btuB product, and the amplified levels of this protein partially reversed the tolerance to E colicins seen in these mutants. J Bacteriol, 1985 Mar, 161(3), 888 - 95 Induction and autoregulation of ada, a positively acting element regulating the response of Escherichia coli K-12 to methylating agents; Lemotte PK et al.; The ada gene of Escherichia coli K-12, the regulatory locus for the adaptive response to methylating agents, coded for a 39,000-dalton protein . An adjacent gene coding for a 27,000-dalton protein was coregulated with ada . The Ada protein was strongly induced upon exposure of cells to methylating agents such as N-methyl-N'-nitro-N-nitrosoguanidine . An analysis of ada regulation with an ada-lacZ operon fusion showed that ada+ function was required for induction of ada transcription . Derivatives of the ada gene truncated from the 3' end produced proteins which were more potent in stimulating transcription than the product of the intact ada gene, indicating that the transcription-activating function of the Ada protein resided in its amino terminus . The sequence of the ada-regulatory region and the identification of the start site of ada transcription are also presented. J Bacteriol, 1985 Mar, 161(3), 1219 - 21 Site-directed insertion and deletion mutagenesis with cloned fragments in Escherichia coli; Winans SC et al.; A mutation of a cloned gene that has been made by introducing a transposon or some other selectable genetic determinant can be crossed into the gene's original replicon by linearizing the cloned DNA and transforming a recB recC sbcB mutant . A number of applications of this method are described with genes of either chromosomal or plasmid origin. J Bacteriol, 1985 Mar, 161(3), 1112 - 7 Excision and reintegration of the Escherichia coli K-12 chromosomal element e14; Brody H et al.; The genetic element e14 is a natural component of the Escherichia coli K-12 chromosome . On induction of the SOS pathways, e14 excises as a 14.4-kilobase circle . We report here on the reintegration of e14 into the chromosome of cured (e14 degrees) E . coli K-12 derivatives . Using a Tn10 insertion mutant of e14, we found that reintegration occurred specifically at the locus originally occupied by e14 and with the same orientation . The reintegration event required neither the RecA nor the RecB functions . The attachment site of the free form was located within a 950-base-pair HindIII-AvaI fragment and shared sufficient homology with the host attachment site to form detectable DNA-DNA hybrids . Even though E . coli C and B/5 did not contain e14, they did possess a HindIII restriction fragment that hybridized to the free e14 attachment fragment . E . coli C could be transformed with e14-1272::Tn10, resulting in integration at this site of homology . The Tn10 mutants were also used in mapping the point of e14 attachment . We found the following sequence: fabD purB atte14 umuC . Furthermore, analysis of a recombinant plasmid that contained both the e14 attachment site and the purB locus showed that these two loci occur within 11 kilobases of each other. Infect Immun, 1985 Mar, 47(3), 808 - 13 Naturally occurring antibodies in human sera that react with the iron-regulated outer membrane proteins of Escherichia coli; Griffiths E et al.; Sera from normal healthy human adults and infants, as well as sera from mice, rabbits, and guinea pigs, were examined by immunoblotting for naturally occurring antibodies reacting with outer membrane proteins of two Escherichia coli strains, O111 and O18 . Some individuals had antibodies reacting very strongly with the iron-regulated outer membrane proteins, including the ferric-enterochelin receptor protein (Mr, 81,000), as well as with ompA . However, sera from infants contained predominantly antibodies to ompA; antibodies recognizing the iron-regulated outer membrane proteins were either absent or barely detectable . In human serum the antibodies were mainly of the immunoglobulin G class . No serotype-specific antibodies to the lipopolysaccharide of E . coli O111 or O18 were found in the sera tested. Biochim Biophys Acta, 1985 Mar 1, 827(3), 472 - 5 Do vanadate polyanions inhibit phosphotransferase enzymes? Boyd DW, Kustin K, Niwa M. Decavanadate inhibits hexokinase, adenylate kinase and phosphofructokinase; neither mono-, tri nor tetrameric vanadate anion is an inhibitor . Decavanadate inhibits phosphofructokinase obtained from bacterial and protistic sources . No form of vanadium(V) anion inhibits galacto-, glycero-, pyruvate and creatine kinase, or inorganic pyrophosphatase . Decavanadate appears to be a non-competitive inhibitor of both hexokinase substrates. J Gen Microbiol, 1985 Mar, 131 ( Pt 3), 571 - 80 Characterization of DNA fragments encoding fimbriae of the uropathogenic Escherichia coli strain KS71; Rhen M; Recombinant plasmids were constructed that expressed the KS71A, KS71B and KS71C fimbrial antigens of the pyelonephritogenic Escherichia coli strain KS71 (O4:K12) in E . coli HB101 . The KS71C-encoding genes were located on a 6.4 kb HindIII-XhoI fragment obtained from the recombinant cosmid pKTH145 that expresses this antigen . Spontaneous KS71C-mutants were isolated that contained a 0.8 kb insert in a specific restriction fragment of KS71C-encoding recombinant plasmids . The KS71B-encoding segment was located on a 11.5 kb deletable DNA fragment of recombinant cosmid pKTH144 . A DNA fragment encoding the KS71A fimbria was obtained on a 12 kb EcoRI fragment of the recombinant cosmid expressing this antigen in E . coli HB101 and closely resembled the KS71B-encoding fragment . In the recombinant cosmid, the KS71B-expressing region was flanked by homologous DNA segments . A similar stretch of DNA was found close to the KS71A-expressing DNA region. Biochem Int, 1985 Mar, 10(3), 385 - 93 Bovine heart mitochondrial F6: HPLC purification, NH2-terminal sequence and the possible structural relatedness to other components of ATPase complexes; Crabb JW et al.; The mitochondrial factor F6 has been purified by reverse-phase HPLC and the molecular weight (8500), amino acid composition and about 25% of the amino acid sequence determined . In the NH2-terminal sequence of the first 18 amino acids (NKELDPVQKLFVDKIREY), six identities with the NH2-terminal sequence of the oligomycin-sensitivity conferring protein (OSCP) are apparent, as well as less striking similarities with the OSCP related subunit delta of E . coli F1 . The possibility that F6, OSCP and subunit delta of E . coli F1 could have evolved from a common ancestral gene is supported by apparent gene duplication within the OSCP and subunit delta sequences. Mol Biol (Mosk), 1985 Mar-Apr, 19(2), 359 - 370 {A model of enzymatic decarboxylation of glutamic acid}; Almazov VP et al.; Interaction of glutamate decarboxylase with its adequate substrate and some quasi-substrates was studied by spectrokinetic, quantum-chemical and some other approaches . It was shown that in the course of decarboxylation an abortive transamination of pyridoxal-5'-phosphate leading to the enzyme inactivation does occur . Identification of intermediate coenzyme-substrate complexes allowed to formulate a model of enzymatic decarboxylation taking into account both the main and abortive reactions . The analysis of electronic structure of the intermediates revealed some of the factors determining the functional specificity of the reaction under study. J Hosp Infect, 1985 Mar, 6 Suppl A, 51 - 7 New methods for determining pre-operative and postoperative skin disinfection; Gundermann KO et al.; A comparison was made of the Thran pressurized spray gun and scrubbing with cotton swabs on the removal of organisms from the skin . Both methods showed similar results but sometimes considerable differences in counts were found on adjacent skin areas . The cotton swab method was used to compare the effect of 60 or 70% isopropanol and 10% povidone-iodine on the flora of the outer side of the upper arm over 24 h . With the exception of one test person, no significant difference was found between the disinfectants. J Hosp Infect, 1985 Mar, 6 Suppl A, 45 - 50 Studies on the use of povidone-iodine with the 'hygienic hand disinfection' test; Newsom SW et al.; The DGHM test for hygienic hand disinfection is based on that of Rotter et al . (1980), and compares the ability of a test agent with 60% isopropanol to reduce Escherichia coli counts on artificially contaminated fingers using 15 volunteers . The test was performed seven times--three with 10% povidone-iodine (PVP-I) scrub, three with 10% PVP-I solution and once with 5% PVP-I solution . Initial tests with PVP-I scrub ('Betadine') gave comparable results with the iso-propanol, but the latter gave an activity below that normally expected, and specified in the Austrian standard (but not in the DGHM test) . A repeat test with greater attention to detail gave similar activity with the scrub, but markedly greater activity from the iso-propanol so that the PVP-I scrub failed the test . Subsequent tests with four aqueous PVP-I solutions showed that all passed the tests with activity marginally greater than that of iso-propanol . Interestingly enough, two German formulations of PVP-I solution showed almost identical activity. Neuroendocrinology, 1985 Mar, 40(3), 193 - 200 Somatostatin release from the median eminence of unanesthetized rats: lack of correlation with pharmacologically suppressed growth hormone secretion; Fukata J et al.; We describe a push-pull perfusion technique to investigate the role of somatostatin in the pharmacological suppression of growth hormone (GH) secretion . Immunoreactive somatostatin (IRS) released from the median eminence (ME) was studied in chronically cannulated, unanesthetized male rats . In control rats which received vehicle injection, plasma GH levels showed a normal ultradian rhythm and relatively stable levels of IRS (around 30 pg/15 min) appeared in the perfusate during the 6-h perfusion . Intracerebroventricular (i.c.v) administration of human growth hormone (40 micrograms), neurotensin (2 micrograms), glucagon (25 micrograms) or intravenous (i.v.) injection of oxymetazoline (50 micrograms/kg b.w.), an alpha-adrenergic agonist, or endotoxin (150 micrograms/kg b.w.) suppressed subsequent GH surges . In these rats, however, IRS levels in the ME perfusate failed to change significantly compared to control rats . These results suggest that changes in somatostatin release may play a minor role in the suppression of plasma GH levels caused by these substances, and that major regulatory effects may be achieved via the suppression of growth hormone-releasing factor release. Mol Biol (Mosk), 1985 Mar-Apr, 19(2), 553 - 7 {Contacts of ribosomal proteins with tRNAPhe and 16S RNA in analogs of the 30S initiation complex}; Abdurashidova GG et al.; Direct RNA-protein contacts have been studied by means of ultraviolet-induced (254 nm) cross-links inside complexes of NAcPhe-tRNAPhe, Phe-tRNAPhe and deacylated tRNAPhe with poly(U)-charged 30S subunit of Escherichia coli ribosome . In the first two complexes tRNA directly contacts with the similar sets of proteins (S4, S5, S7, S9/S11; S6 and S8 are found only in the second complex) . These sets are similar to that in the fMet-tRNAfMet X 30S X mRNA complex, evidencing similar disposition of tRNAs in these three complexes . 16S RNA contacts in free 30S subunit mainly with proteins S4, S7 and S9/S11 . In both complexes, containing NAcPhe-tRNAPhe and Phe-tRNAPhe, 16S RNA contacts with essentially the same proteins (S4, S5, S7, S8, S9/S11, S10, S15, S16 and S17) and in the same ratio, evidencing similar conformation of 30S subunit in these two complexes . In the third complex deacylated tRNAPhe contacts with proteins S4, S5, S6, S8, S9/S11 and S15, 16S RNA-protein interaction differs from those in the first two complexes by a remarkable decrease of cross-linked proteins S8, and S9/S11 and by the appearance of a large amount of cross-linked proteins(s) S13/S14 . Hence, this complex differs from the first two by conformation of 30S subunit and, probably, by disposition and/or conformation of tRNA. Mol Biol (Mosk), 1985 Mar-Apr, 19(2), 371 - 7 {The peptidyltransferase center of ribosomes--what is it?}; Kukhanova MK et al.; The recent data on the interaction of model substrates and substrate-like analogs with acceptor and donor sites of 70S and 80S ribosomes are considered in terms of peptidyl transferase center models suggested earlier. J Bacteriol, 1985 Mar, 161(3), 1162 - 70 Escherichia coli 6S RNA gene is part of a dual-function transcription unit; Hsu LM et al.; The gene coding for the metabolically stable 6S RNA of Escherichia coli has been cloned, sequenced, and partially characterized in expression analyses . The DNA sequence results confirm the accuracy of the previously established RNA sequence and, with genomic hybridization data, reveal that there is only one copy of the 6S DNA in the chromosome . Consistent with its relaxed mode of expression, the promoter region of the 6S RNA gene was found to lack the hypothetical GC-rich discriminator domain common to other stable RNA genes under stringent control . The sequence results also revealed the occurrence of a 540-base-pair open reading frame immediately downstream from the 6S RNA coding region . Results from the expression analyses show that the protein and RNA coding regions are cotranscribed in vitro and that the open reading frame is translated in vivo. J Bacteriol, 1985 Mar, 161(3), 1156 - 61 Escherichia coli 6S RNA is not essential for growth or protein secretion; Lee CA et al.; The function of the stable 6S RNA of Escherichia coli is not known . Recently, it was proposed that the 6S RNA is a component of a bacterial signal recognition particle required for protein secretion . To test this proposal, we isolated a mutant that lacks the 6S RNA . Studies of the mutant show that the 6S RNA is not essential for growth or for protein secretion . The gene for the 6S RNA (ssr) maps near serA at 63 min on the E . coli genetic map. Cell, 1985 Mar, 40(3), 527 - 35 Control of ColE1 plasmid replication: initial interaction of RNA I and the primer transcript is reversible; Tomizawa J; Replication of ColE1-type plasmids is known to be regulated by a plasmid-specific RNA (RNA I), whose binding to the transcript (RNA II) from the primer promoter results in inhibition of formation of the primer for DNA replication . In this paper, it is shown that binding of RNA I to the homologous RNA II is inhibited by an RNA I specified by a plasmid of different compatibility . The inhibition is caused by the reversible interaction of RNA II with the heterologous RNA I, which competes with reversible interaction of the two homologous RNAs at a step preceding their stable binding . As predicted from these results, the copy numbers of both ColE1 and RSF1030 are increased when both plasmids are present in the same cell . The Rom protein enhances the reversible interaction. Biochimie, 1985 Mar-Apr, 67(3-4), 335 - 42 Mutagenesis and growth delay induced in Escherichia coli by near-ultraviolet radiations; Favre A et al.; The literature relating to genetic changes induced in Escherichia coli by near-ultraviolet radiations is reviewed and summarized: i) these radiations are much less mutagenic than would be expected from the known level of DNA damage, ii) pre-illumination with near-UV light antagonizes the mutagenic effect of UV (254 nm) light . In agreement with these findings, the SOS functions are not induced by near-UV radiations . Furthermore prior exposure of cells to near-UV light inhibits the subsequent 254 nm induction of the SOS response . Among the several hypothesis considered to explain these observations, one can be clearly favoured . Near-UV light triggers, at sublethal fluences, the growth delay effect . The target molecules, tRNAs, are photocrosslinked and some tRNA species become poor substrates in the acylation reaction . In vivo these tRNA molecules accumulate on the uncharged form, leading to a transient cessation of protein synthesis . The SOS response is inducible and as such requires protein synthesis . We therefore propose that near-ultraviolet radiations have a dual effect: i) they induce, mostly indirectly, DNA lesions which are potentially able to trigger the SOS response, ii) they prevent the expression of the SOS functions through the transient inhibition of protein synthesis (growth delay). Ann Trop Paediatr, 1985 Mar, 5(1), 19 - 22 Enteropathogenic Escherichia coli (EPEC) and enterotoxigenic (ETEC) related diarrhoeal disease in a neonatal unit; Adhikari M et al.; In an outbreak of summer diarrhoea in the neonatal unit, King Edward VIII Hospital, Durban, 25 (69%) of the 36 infants had organisms demonstrated in their stools . Four (11%) had EPEC alone, six (17%) ETEC alone, six (17%) EPEC plus rotavirus and nine (25%) all three organisms . Eleven (30%) infants had no organisms in their stools . Rotavirus alone was not present in any of the stools . Seven infants had septicaemia . The overall mortality was 22% and 62.5% of the deaths occurred in low birthweight infants . In a study of 41 infants without diarrhoea during the following winter and summer periods 55% of winter, 43% of summer controls, and four of 12 (33%) mothers had rotavirus . Only two (4.8%) of 41 infants had E . coli (EPEC) . The findings suggest that E . coli (EPEC strain 044/K74{c}, and ETEC) was the major cause of the outbreak and it was associated with a high mortality. EMBO J, 1985 Mar, 4(3), 699 - 706 Precise epitope mapping of the murine transformation-associated protein, p53; Wade-Evans A et al.; Murine p53 cDNA sequences were cloned into an in vitro expression vector, Protem Hind . Four deletion libraries were generated using Bal31 double-stranded exonuclease; two being made from constructs encoding a fusion protein constructed from SV40 small t sequences and the p53 clone, p27.la; and two from the full length p53 clone, pp53-5 . Both 5'- and 3'-terminal deletions of the p53 gene were made . Transcription of these constructs using Escherichia coli RNA polymerase holoenzyme, followed by translation in mRNA-dependent rabbit reticulocyte lysate, gave in vitro, truncated protein products which were immunoprecipitated by a panel of anti-p53 monoclonal antibodies . This approach enabled us to map accurately the binding sites of seven different monoclonal antibodies, demonstrating four distinct antigenic sites on p53 . A synthetic peptide was constructed corresponding to the predicted amino acid sequence of one of these epitopes . This peptide competes with the epitope on the full length p53 protein for the relevant monoclonal antibodies and dissociates the corresponding p53/antibody complexes. Bioorg Khim, 1985 Mar, 11(3), 417 - 9 {Changes in subunit conformation and their reciprocal configuration in the transition from the pretranslocated to the posttranslocated state}; Abdurashidova GG et al.; RNA-protein contacts in pretranslocated and posttranslocated states of E . coli ribosomes have been determined by means of UV-induced cross-linking . In the two functional states as well as in free 70C ribosome, the same proteins are involved in RNA-protein intersubunit contacts, located in the region of L1 protuberance (left side of 70S ribosome) . The transition from pre- to posttranslocated state is accompanied by disappearance of RNA-protein contacts in the region of L7/L12 stalk . This favours the locking-unlocking model of the translating ribosome. Res Vet Sci, 1985 Mar, 38(2), 246 - 7 Serological comparison of the Escherichia coli prototype strains for the F(Y) and Att 25 adhesions implicated in neonatal diarrhoea in calves; Morris JA et al.; Slide agglutination tests using single absorbed and double absorbed antisera indicated that the Att 25 prototype Escherichia coli strain 25 KH9 produces the F(Y) adhesion; that this E coli also produces at least one other surface antigen not found on the F(Y) prototype E coli strain 11a; and that F(Y)+ E coli strain 28a produces at least one other surface antigen not produced by the prototype strains for the F(Y) and Att 25 antigens . These antigens were found on E coli isolated from outbreaks of calf diarrhoea in the United Kingdom. Biochem Biophys Res Commun, 1985 Feb 28, 127(1), 31 - 6 High-affinity calcium-binding proteins in Escherichia coli; Harmon AC et al.; Crude extracts of Escherichia coli contain at least three heat stable proteins of Mr, 33,000, 47,000, and 60,000, which bind 45Ca2+ in buffers containing micromolar calcium and physiological salt concentrations . Fractions containing these proteins neither activated the calmodulin-dependent enzyme, NAD kinase, nor inhibited the activity of this enzyme in the presence of brain calmodulin . Radioimmunoassay of crude extracts for calmodulin indicated the presence of a calmodulin-like antigen . Crude extracts also contain proteins that interact with 2-trifluoromethyl-10H-(3'-aminopropyl)phenothiazine-Sepharose in a calcium-dependent manner, but proteins eluted from this resin did not bind calcium with high affinity. Biochemistry, 1985 Feb 26, 24(5), 1175 - 80 Methionyl-tRNA synthetase from Escherichia coli: primary structure at the binding site for the 3'-end of tRNAfMet; Hountondji C et al.; It was previously shown that when the tryptic fragment of methionyl-tRNA synthetase from Escherichia coli is incubated with periodate-treated initiator tRNA, it is inactivated due to the formation of a covalent 1:1 complex that could be stabilized by reduction with cyanoborohydride {Hountondji, C., Fayat, G., & Blanquet, S . (1979) Eur . J . Biochem . 102, 247-250} . In this work, the residues labeled in the trypsin-modified enzyme have been identified . After chymotryptic digestion of the protein-tRNA complex, two major labeled peptides (A and B) and a minor one (C) were isolated and identified by sequencing . The radioactivity associated with peptides A-C represented 65-75, 20-25, and 2-4%, respectively, of the radioactivity eluted from the peptide maps . Peptides A and B encompassed lysines-335 and -61, respectively . Both these lysines were fully labeled . Peptide C encompassed lysines-142, -147, and -149, each of which was incompletely labeled . The significance of these results is discussed in light of the known crystallographic structure of the enzyme. Biochemistry, 1985 Feb 26, 24(5), 1116 - 21 Chorismate mutase-prephenate dehydrogenase from Escherichia coli: positive cooperativity with substrates and inhibitors; Christopherson RI et al.; Investigations have been made at pH 6.0 of the effect of chorismate and adamantane derivatives on the mutase and dehydrogenase activities of hydroxyphenylpyruvate synthase from Escherichia coli . When used over a wide range of concentrations, chorismate 5,6-epoxide, chorismate 5,6-diol, adamantane-1,3-diacetate, adamantane-1-acetate, adamantane-1-carboxylate, and adamantane-1-phosphonate give rise to nonlinear plots of the reciprocal of the initial velocity of each reaction as a function of the inhibitor concentration . The inhibitors do not induce the enzyme to undergo polymerization and have only a small effect on the S20,w value of the enzyme as determined by using sucrose density gradient centrifugation . At low substrate concentration, low concentrations of adamantane-1-acetate cause activation of both the mutase and dehydrogenase activities while at higher concentrations this compound functions as an inhibitor . When chorismate and prephenate are varied over a wide range of concentrations, double-reciprocal plots of the data indicate that the reactions exhibit positive cooperativity . The addition of albumin eliminates the cooperative interactions associated with substrates but has little effect on those associated with inhibitors. Biochemistry, 1985 Feb 26, 24(5), 1221 - 6 Comparative study of glutamine synthetase bound lanthanide(III) ions using NMR relaxation and lanthanide(III) luminescence techniques; Eads CD et al.; Changes in the intrinsic fluorescence intensity of glutamine synthetase induced by lanthanide(III) ion binding demonstrate the existence of three types of sites for these ions . The sites are populated sequentially during titrations of the enzyme, and the first two have a stoichiometry of 1 per enzyme subunit . The number of water molecules coordinated to Eu(III) bound to the first site was determined by luminescence lifetime techniques to be 4.1 +/- 0.5 . The hydration of Gd(III) bound to the same site was studied by magnetic field dependent water proton longitudinal relaxation rate measurements, and by water proton and deuteron relaxation measurements of one sample at single magnetic fields . The magnetic resonance techniques also yield a value of 4 for the hydration number. Nucleic Acids Res, 1985 Feb 25, 13(4), 1163 - 72 The mac promoters: functional hybrid promoters activated by the malT product and repressed by the lacI product; Vidal-Ingigliardi D et al.; Using in vitro techniques we have fused upstream sequences from the malPp promoter (normally activated by the MalT protein) to downstream sequences from the lacZp promoter (normally repressed by the LacI protein) . Several hybrid promoters were thus obtained, which were controlled by the MalT protein, but were poorly active . More efficient promoters were then isolated using in vivo selection . Three main conclusions could be derived from the analysis of all of these hybrid promoters . Firstly, the MalT protein seems able to force RNA polymerase to start transcription at any DNA sequence, albeit with a low efficiency . Secondly, the strength of the hybrid promoters is considerably increased if a Pribnow Box is positioned at a precise location with respect to the MalT binding site . Thirdly, the presence of the lac operator, even when properly positioned with respect to the transcription startpoint, does not suffice to permit full repression by the lacI product. J Biol Chem, 1985 Feb 25, 260(4), 2226 - 30 Two mutations in the dispensable part of alanine tRNA synthetase which affect the catalytic activity; Jasin M et al.; Two previously described chromosomal mutant alleles, alaS4 and alaS5, of Escherichia coli Ala-tRNA synthetase have been analyzed . Each causes a sharp diminution in aminoacylation activity and disrupts the alpha 4 tetramer structure of identical chains of 875 amino acids; neither mutation significantly disturbs the activity for synthesis of alanyladenylate . The location of each mutation within the structural gene has been mapped by marker rescue with specific gene fragments . Each mutant allele was cloned from the genome by reciprocal recombination with a multicopy plasmid that contains segments of alaS which flank the respective mutations . Further analysis established: 1) a single G----A transition results in a Gly----Asp change for each mutant allele at codon 674 (alaS4) and at codon 677 (alaS5) . 2) The mutations are in the oligomerization domain, about 200 amino acids beyond the C-terminal side of the catalytic domain that previously was mapped by deletion analysis; the mutations are, thus, in a part of the polypeptide which is dispensable for catalytic activity . 3) For both mutant enzymes, there is little effect of the mutation on the Km for tRNAAla; kcat for aminoacylation is decreased by an order of magnitude . These point mutations reveal a subtle integration of the catalytic core with parts of the polypeptide that are not essential for catalytic activity. J Biol Chem, 1985 Feb 25, 260(4), 2077 - 9 Reaction of substrates with 35S-thiophosphorylated succinyl-CoA synthetase of pig heart . Similarities to the case of the Escherichia coli enzyme; Nishimura JS et al.; Guanosine 5'-O-(3-thio)triphosphate (GTP gamma S) was found to be a substrate of pig heart succinyl-CoA synthetase with Km and kcat values of 3 microM and 0.23 s-1, respectively . The corresponding values with GTP as substrate were 48 microM and 65 s-1 . 35S-thiophosphorylated enzyme was prepared by incubation of pig heart succinyl-CoA synthetase with {35S}GTP gamma S . A comparison was made of thiophosphoryl group release by substrates from this alpha beta (one active site) enzyme with that of the alpha 2 beta 2 (two active sites) Escherichia coli enzyme (Wolodko, W . T., Brownie, E . R., O'Connor, M . D., and Bridger, W . A . (1983) J . Biol . Chem . 258, 14116-14119; Nishimura, J . S., and Mitchell, T . (1984) J . Biol . Chem . 259, 9642-9645) . It was found, as in the case of the E . coli enzyme, that thiophosphoryl group release by GDP and by succinate plus CoA was stimulated by succinyl-CoA and GTP, respectively . The same result was observed at 1, 0.1, and 0.01 mg/ml, lending assurance that these phenomena were not exhibited by an aggregated form of the pig heart enzyme . While an alternating-sites catalytic cooperativity model is not ruled out for the E . coli enzyme, it is proposed that the NTP- and succinyl-CoA-stimulated release of thiophosphoryl groups from either enzyme involves a "same-site" mechanism, to be distinguished from an "other-site" mechanism. FEBS Lett, 1985 Feb 25, 181(2), 407 - 11 Convenient modification of the method for oligonucleotide-directed in vitro mutagenesis of cloned DNA; Efimov VA et al.; A new modification of the oligonucleotide-mediated mutagenesis technique has been developed . The proposed methodology has been used to produce specific base changes in the double-stranded plasmid DNA . For this purpose, special cloning vectors have been constructed using the synthetic oligodeoxyribonucleotides . The developed method allows the production of mutant DNA from those of the wild-type with a yield of 10-20%. FEBS Lett, 1985 Feb 25, 181(2), 236 - 40 Plasmid-encoded initiation protein is required for activity at all three origins of plasmid R6K DNA replication in vitro; Inuzuka M; DNA replication of plasmid R6K initiates at three unique sites, ori alpha, ori beta, and ori gamma . Replicating DNA molecules of a deletion derivative of R6K were synthesized in an in vitro system containing pi protein fraction from cells carrying a mini-R6K derivative that produced only this initiation protein as an R6K-encoded protein and analyzed by electron miscroscopy . Requirement of pi protein for the activity of all these three replication origins in vitro was verified . Frequencies of initiation at the three origins were almost equal. Nucleic Acids Res, 1985 Feb 25, 13(4), 1185 - 92 Escherichia coli 23S ribosomal RNA truncated at its 5' terminus; Sirdeshmukh R et al.; In a strain of E . coli deficient in RNase III (ABL1), 23S rRNA has been shown to be present in incompletely processed form with extra nucleotides at both the 5' and 3' ends (King et al., 1984, Proc . Natl . Acad . Sci . U.S . 81, 185-188) . RNA molecules with four different termini at the 5' end are observed in vivo, and are all found in polysomes . The shortest of these ("C3") is four nucleotides shorter than the accepted mature terminus . In growing cells of both wild-type and mutant strains up to 10% of the 23S rRNA chains contain the 5' C3 terminus . In stationary phase cells, the proportion of C3 termini remains the same in the wild-type cells; but C3 becomes the dominant terminus in the mutant . Species C3 is also one of the 5' termini of 23S rRNA generated in vitro from larger precursors by the action of purified RNase III . We therefore suggest that some form of RNase III may still exist in the mutant; and since no cleavage is detectable at any other RNase III-specific site, the remaining enzyme would have a particular affinity for the C3 cleavage site, especially in stationary phase cells . We raise the question whether the C3 terminus has a special role in cellular metabolism. J Biol Chem, 1985 Feb 25, 260(4), 1987 - 90 Purification and crystallization of the EcoRV restriction endonuclease; D'Arcy A et al.; The type II restriction endonuclease EcoRV purified from a genetically engineered, overproducing strain has been crystallized . Four crystal forms all obtained by precipitation with polyethylene glycol 4000 have been characterized . Two of these are suitable for high resolution structure analysis . Both are orthorhombic, have space group P2(1)2(1)2(1) and have similar unit cell dimensions of a = 58.2 A, b = 71.7 A, c = 130.6 A (form A) and a = 59.9 A, b = 74.5 A, c = 121.8 A (form B) . They diffract to about 2A resolution and appear to have one dimer of 2 X 29,000 daltons in the asymmetric unit. Nucleic Acids Res, 1985 Feb 25, 13(4), 1303 - 16 Nucleotide sequence of the guaB locus encoding IMP dehydrogenase of Escherichia coli K12; Tiedeman AA et al.; IMP dehydrogenase, the product of the guaB locus in Escherichia coli K12, catalyzes the synthesis of XMP by the NAD+ dependent oxidation of IMP . The guaB locus has been subcloned from the Clarke and Carbon plasmid pLC34-10 . The sequence of the guaB structural gene and surrounding DNA was determined by the dideoxy chain termination method of Sanger . The 1.533 kb guaB gene encodes an IMP dehydrogenase subunit of molecular weight 54,512 . S1 nuclease mapping placed the site of guaBA mRNA initiation approximately 188 bp from the start of the guaB structural gene . The -10 and -35 regions that define the guaBA promoter were located upstream of the start of the guaBA transcription initiation site . The control region of approximately 188 bp does not show any obvious potential for secondary structure . A secondary lambda att site has been identified 42 bp distal to the guaB start codon. FEBS Lett, 1985 Feb 25, 181(2), 367 - 72 The effect of GTP hydrolysis and transpeptidation on the arrangement of aminoacyl-tRNA at the A-site of Escherichia coli 70 S ribosomes; Vladimirov SN et al.; From the affinity labelling of 70 S ribosomes with a photoreactive derivative of Phe-tRNAPhe bearing an arylazido group on guanine residues, it has been found that different sets of ribosomal proteins are labelled in the course of three successive steps of EF-Tu-dependent binding of aminoacyl-tRNA derivative at the A-site . Proteins S5, S7, S8, S16, S17, L9, L14, L15 and L24 were labelled before GTP hydrolysis; proteins S5, S7, S9, S11, S14, S18, S19, S21, L9, L21 and L29--after GTP hydrolysis; proteins S2, S5, S7, S21, L11 and L23--after GTP hydrolysis and transpeptidation. J Mol Biol, 1985 Feb 20, 181(4), 467 - 78 Attenuation control of the Escherichia coli phenylalanyl-tRNA synthetase operon; Springer M et al.; The pheST operon codes for the two subunits of the essential enzyme phenylalanyl-tRNA synthetase . The nucleotide sequence of the regulatory regions of the operon, in vitro transcription data and in vivo experiments indicate that the operon is controlled by attenuation in a way similar to many amino acid biosynthetic operons . In this work the control of the pheST operon was studied in vivo by measuring the effect of deletions in the regulatory regions on downstream expression . The presence of a strong promoter followed by an approximately 90% efficient terminator in front of the structural parts of the operon is demonstrated . An open reading frame coding for a 14 amino acid long leader peptide containing five phenylalanine residues is located between the promoter and the terminator . The presence of the transcription terminator is shown to be essential to the operon's regulation . The localization of the promoter and the terminator agrees with the results of previous in vitro experiments . It is also shown that about 30% of the transcripts covering the pheST operon come from the upstream gene, rplT, which codes for the ribosomal protein L20 . Although cotranscription exists between rplT and pheST, these genes are not systematically coregulated since reducing the translation of rplT about tenfold, does not change pheST expression . The pheST operon is also shown to be derepressed by a cellular excess of phenylalanyl-tRNA synthetase . This derepression is shown to be due to the pheST attenuator. J Mol Biol, 1985 Feb 20, 181(4), 551 - 5 A frameshift mutation at the junction of an IS1 insertion within lacZ restores beta-galactosidase activity via formation of an active lacZ-IS1 fusion protein; Malamy MH et al.; The insertion of IS1 elements into lacZ results in the loss of beta-galactosidase activity, and such insertions exert a severe polar effect on the expression of the distal genes of the operon . In addition to these properties, the mutation lacZ::IS1-MS319 has the unique property of reversion to Lac+ (ts) spontaneously or after treatment with the frameshift mutagen ICR-191; such revertants retain the IS1 element . We have determined that the site of integration of IS1 into lacZ is at position 4338, 18 nucleotides from the end of the sequence encoding the C-terminus of beta-galactosidase . Reversion to Lac+ promoted by ICR-191 results from the loss of a G residue from a GGG sequence located at the junction of lacZ and IS1 . As a result an active, but temperature-sensitive, lacZ-IS1 fusion protein is formed containing six amino acids derived from IS1 which replace six amino acids encoded by lacZ . The IS1 element in MS319 is a new member of the iso-IS1 family, which we designate IS1T. J Mol Biol, 1985 Feb 20, 181(4), 545 - 50 Mapping of DNA gyrase cleavage sites in vivo oxolinic acid induced cleavages in plasmid pBR322; O'Connor MB et al.; We have developed a procedure which permits the mapping of DNA gyrase cleavage sites in vivo . Addition of oxolinic acid, an inhibitor of DNA gyrase, to growing cells of Escherichia coli containing the plasmid pBR322 resulted in double-strand cleavage of DNA, and allowed the isolation of significant quantities of linearized plasmid DNA after lysis of treated cells with sodium dodecyl sulfate . Initially the linear product was purified from agarose gels, cleaved by restriction endonucleases, and then subjected to Southern hybridization analysis using defined DNA probes . A number of distinct cleavage sites, used with varying degrees of efficiency, were identified within pBR322 using this simple procedure . To achieve greater resolution and to improve sensitivity, we then employed an electroblotting procedure to transfer DNA fragments from acrylamide gels onto nylon membranes . This alternative method does not require the isolation of the linearized product before performing the mapping procedure . The improved resolution obtained from acrylamide gels and the superior binding properties of the nylon membranes have allowed us to accurately map 74 distinct oxolinic acid-induced cleavage sites within pBR322 . The significance of these findings in light of previously reported studies in vitro, as well as the possible role of such sites during illegitimate recombination, are discussed. Biochim Biophys Acta, 1985 Feb 20, 824(2), 121 - 7 Verification of a new model of the time course of RNA synthesis . Measurement of the rates of initiation and elongation; McWilliam P et al.; The time course of RNA synthesis in vitro commonly starts with a lag followed by a linear phase . Differing from the earlier interpretation we have previously proposed that, under conditions where the initiation rate is low, the lag represents the time taken for the first RNA polymerase molecule to reach a termination site . During the linear phase, initiation is balanced by termination (Mahon, G.A.T., McWilliam, P., Gordon, R.L . and McConnell, D.J . (1980) J . Theor . Biol . 87, 483-515) . We report the use of rifampicin as a further test of this new model . We show that it does apply under conditions of high ionic strength (0.3 M KCl), and under these conditions time courses may be analyzed to yield unbiased estimates of the initiation (Vi) and chain elongation (Vp) rates . We illustrate the application of the method of time course analysis and confirm some of its features by examining the effect of variation in the concentrations of RNA polymerase and nucleoside triphosphate on the estimates of Vi and Vp . The alternative interpretation of the time course applies under conditions of low ionic strength, where the initiation rate is high . (Chamberlin, M.J., Nierman, W.C., Wiggs, J . and Neff, N . (1979) J . Biol . Chem . 254, 10061-10069.) The advantages of each model in measuring Vi and Vp (the major parameters of the transcription reaction) are discussed. Biochem J, 1985 Feb 15, 226(1), 217 - 23 The purification of shikimate dehydrogenase from Escherichia coli; Chaudhuri S et al.; A procedure was developed for the purification of shikimate dehydrogenase from Escherichia coli . Homogeneous enzyme with specific activity 1100 units/mg of protein was obtained in 21% overall yield . The subunit Mr estimated by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate was 32 000 . The native Mr, estimated by gel-permeation chromatography on a TSK G2000SW column, was also 32 000 . E . coli shikimate dehydrogenase is therefore a monomeric NADP-linked dehydrogenase. Eur J Biochem, 1985 Feb 15, 147(1), 163 - 70 Quaternary structure of chloroplast F1-ATPase in solution . Conformational changes in spatial arrangement of subunits upon activation; Wagner R et al.; The hydrodynamic properties of isolated ATPases were studied via their rotational diffusion in buffer solution . Chloroplast F1-ATPase (CF1) and Escherichia coli F1-ATPase (EF1) were covalently labeled with eosinisothiocyanate and then investigated by polarized laser spectroscopy . The rotational correlation time in aqueous buffer of latent (five-subunit) CF1 was 390 ns . Four-subunit (delta-deficient) CF1 showed the same correlation time, however, for three-subunit (delta, epsilon-deficient) CF1 the rotational correlation time was more than eight times larger (3200 ns) . The rotational correlation time of activated CF1 was three times larger than the one of latent CF1 . These large changes in the rotational correlation times are directly related to changes in the quaternary structure of CF1 upon activation . EF1 was found to behave essentially as activated CF1 . Based on the observed rotational correlation times we concluded that the mass distributions of latent CF1 and of delta-deficient CF1 resemble a dimeric arrangement . The structure of delta, epsilon-deficient CF1 more likely resembles a hexagon, the mass centers of the six main subunits lie in one plane . The structure of the activated forms of CF1 can be described best as an intermediate between the dimeric arrangement of latent CF1 and an octahedron . The large changes in the quaternary structure of isolated CF1 are reversed when the activation of the enzyme is reversed. Arch Biochem Biophys, 1985 Feb 15, 237(1), 217 - 23 Synthesis of the alpha and beta subunits of coupling factor 1 by polysomes from pea chloroplasts; Bhaya D et al.; Washed thylakoids of pea chloroplasts, containing tightly bound polysomes, incorporate radioactive amino acids into protein when supplied with soluble factors from Escherichia coli . Polyacrylamide gel electrophoresis with lithium dodecyl sulfate, followed by autoradiography of the labeled products, showed the synthesis of a number of different polypeptides . Two of the most heavily labeled products were in the region expected for the alpha and beta subunits of coupling factor 1, at 57 and 54 kDa . Positive identification of the subunits was made using monospecific antibodies . Furthermore, the same two polypeptides made by soluble polysomes located in the chloroplast stroma were found . While the major proportion of the newly formed alpha and beta subunits made by thylakoid-bound polysomes remained with the thylakoids after protein synthesis occurred, no evidence was found of incorporation into complete, EDTA-extractable coupling factor 1. Biochem Biophys Res Commun, 1985 Feb 15, 126(3), 1259 - 68 Cloning and expression of the cDNA coding for aequorin, a bioluminescent calcium-binding protein; Prasher D et al.; Aequorin is a bioluminescent protein which consists of a polypeptide chain (apoaequorin), coelenterate luciferin, and bound oxygen . Aequorin produces blue light upon binding Ca2+ . We have isolated six recombinant pBR322 plasmids which contain apoaequorin cDNA sequences . A mixed synthetic pBR322 plasmids which contain apoaequorin cDNA sequences . A mixed synthetic oligonucleotide probe was used to identify these cDNAs . An extract of an E . coli strain possessing the largest cDNA contained apoaequorin . This apoaequorin can be converted to aequorin in the presence of coelenterate luciferin, 2-mercaptoethanol, and O2 . This cDNA is therefore apparently full-length. Biochemistry, 1985 Feb 12, 24(4), 817 - 22 Nuclear magnetic resonance observation and dynamics of specific amide protons in T4 lysozyme; Griffey RH et al.; We have produced T4 lysozyme using a bacterial expression system which allows efficient incorporation of isotopically labeled amino acids in lysozyme . By using conditions that repress the expression of various transaminases, we have incorporated 15N-labeled amino acid into the five phenylalanine residues of the protein . The relatively large spin--spin coupling (87 +/- 3 Hz) between the 15N nucleus and the phenylalanine amide protons may then be exploited in a variety of ways to selectively observe the five phenylalanine amide proton resonances . These include a simple "echo difference" technique which displays the amide proton resonances in one dimension and a "forbidden echo" technique {Bax, A., Griffey, R . H., & Hawkins, B.L . (1983) J . Magn . Reson . 55, 301-335} which gives two-dimensional information allowing the proton and 15N chemical shifts of each amide to be determined . With these approaches, all five phenylalanine amide protons give resolved resonances . Deuterium exchange experiments demonstrate that three of the five resonances are slow to exchange (half-times of about 1 week at pH 5.5 and 4 degrees C) while the other two are rapid with complete exchange in hours or less . These observations correlate well with the secondary structure of the protein which shows three residues in alpha-helical regions and two residues in surface-exposed environments . This approach of isotopic substitution on nitrogen or carbon atoms is of general utility and should allow virtually any proton on a protein of molecular weight 20 000 or thereabout to be selectively observed. Biochemistry, 1985 Feb 12, 24(4), 914 - 22 Catabolism of bis(5'-nucleosidyl) oligophosphates in Escherichia coli: metal requirements and substrate specificity of homogeneous diadenosine-5',5'''-P1,P4-tetraphosphate pyrophosphohydrolase; Plateau P et al.; Diadenosine-5',5'''-P1,P4-tetraphosphate pyrophosphohydrolase (diadenosinetetraphosphatase) from Escherichia coli strain EM20031 has been purified 5000-fold from 4 kg of wet cells . It produces 2.4 mg of homogeneous enzyme with a yield of 3.1% . The enzyme activity in the reaction of ADP production from Ap4A is 250 s-1 {37 degrees C, 50 mM tris(hydroxymethyl)aminomethane, pH 7.8, 50 microM Ap4A, 0.5 microM ethylenediaminetetraacetic acid (EDTA), and 50 microM CoCl2} . The enzyme is a single polypeptide chain of Mr 33K, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis and high-performance gel permeation chromatography . Dinucleoside polyphosphates are substrates provided they contain more than two phosphates (Ap4A, Ap4G, Ap4C, Gp4G, Ap3A, Ap3G, Ap3C, Gp3G, Gp3C, Ap5A, Ap6A, and dAp4dA are substrates; Ap2A, NAD, and NADP are not) . Among the products, a nucleoside diphosphate is always formed . ATP, GTP, CTP, UTP, dATP, dGTP, dCTP, and dTTP are not substrates; Ap4 is . Addition of Co2+ (50 microM) to the reaction buffer containing 0.5 microM EDTA strongly stimulates Ap4A hydrolysis (stimulation 2500-fold) . With 50 microM MnCl2, the stimulation is 900-fold . Ca2+, Fe2+, and Mg2+ have no effect . The Km for Ap4A is 22 microM with Co2+ and 12 microM with Mn2+ . The added metals have similar effects on the hydrolysis of Ap3A into ADP + AMP . However, in the latter case, the stimulation by Co2+ is small, and the maximum stimulation brought by Mn2+ is 9 times that brought by Co2+ . Exposure of the enzyme to Zn2+ (5 microM), prior to the assay or within the reaction mixture containing Co2+, causes a marked inhibition of Ap4A hydrolysis.(ABSTRACT TRUNCATED AT 250 WORDS) FEBS Lett, 1985 Feb 11, 181(1), 138 - 42 Essential structure for full enterotoxigenic activity of heat-stable enterotoxin produced by enterotoxigenic Escherichia coli; Yoshimura S et al.; Several analogues of heat-stable enterotoxins (STh and STp) produced by enterotoxigenic Escherichia coli were synthesized . Peptides (STh{6-18} and STp{5-17}) consisting of 13 amino acid residues from the Cys residue near the N-terminus to the Cys residue near the C-terminus and linked by three disulfide bonds had the same biological and immunological properties as native STh and STp, respectively . The results indicated that the sequence with the 13 amino acid residues and three disulfide linkages is essential for full biological activity of ST. Nucleic Acids Res, 1985 Feb 11, 13(3), 825 - 39 Characterization of a mouse interferon gene locus II . Differential expression of alpha-interferon genes; Kelley KA et al.; A cluster of four MuIFN-alpha genes was recently isolated and characterized (1); one of the genes in this cluster had, in the coding region, an internal deletion of 5 amino acids . Bacterial expression plasmids were constructed to examine the effect of this deletion on the antiviral activity of the MuIFN-alpha 4 peptide and it was found that the alpha 4 interferon peptide had a 100-fold lower antiviral activity than full length alpha-interferon proteins when expressed in E . coli . Three of the four MuIFN-alpha genes identified were expressed coordinately in L-cells infected with NDV . The relative levels of alpha 4 mRNA were substantially higher than the levels of the other alpha mRNAs . Comparison of the 5' end flanking sequences of these four alpha interferon genes revealed that the promoter sequences of alpha 1, alpha 5 and alpha 6 are more homologous to each other than to the alpha 4 promoter which also contains a G rich cluster not seen in the other three promoters. FEBS Lett, 1985 Feb 11, 181(1), 133 - 7 Variability in the nucleic acid binding site size and the amount of single-stranded DNA-binding protein in Escherichia coli; Bobst EV et al.; The Escherichia coli single-stranded DNA binding protein (SSB), essential for DNA replication, recombination and repair, can undergo a thermally induced irreversible conformational change which does not eliminate its biological activity, but changes the number of nucleotides it covers (binding site size) when binding to a single-stranded nucleic acid lattice . The binding site size of native and conformationally changed SSB was also found to be a function of the molecular mass of the polynucleotide, an observation which is unusual for single-stranded DNA binding proteins and will greatly affect the affinity relationship of this protein for nucleic acids . A radioimmunoassay used to quantitate in SSB level in cells revealed the number of SSB tetramers to be larger than initial estimates by a factor of as much as six . All these data suggest that the biological role of SSB and