J Cell Sci, 1985 Oct, 78, 23 - 48 Effect of Cerophyl growth medium on exocytosis in Tetrahymena thermophila; Pesciotta DM et al.; Culturing the ciliate Tetrahymena thermophila in Cerophyl has provided an opportunity for studying the assembly and/or synthesis of the intramembrane particle array, the rosette, which marks the site of exocytosis in these cells . Cultures grown in this medium cease cell division after only 12h and enter 'stationary phase' earlier (12h of growth) relative to growth in standard medium (proteose peptone) . In addition, the cell changes from the normally observed pear-shaped body to a thinner more ellipsoid form . Despite the initial similarities to starving cells, several differences are observed in the Cerophyl-grown cells . One is that cell size remains constant for at least 72h in contrast to starved cells . Secondly, in spite of this block in cell division, results from freeze-fracture replicas of the cell membrane of these cells show that they continue to assemble rosettes, the number of which increases approximately six times, from 0.34 rosette/microgram2 to 2.1 rosettes/microgram2 . Addition of the protein synthesis inhibitor, cycloheximide (6h exposure), during growth in Cerophyl shows that 70% of rosettes can be assembled, despite the blockage of translation, by using pre-existing component(s) from a pool . The remaining 30% must involve de novo synthesis of one or more components; this percentage can be increased with longer exposure to the drug . Thirdly, an apparent increase in the number of mucocysts is observed by thin-section electron microscopy . At first (12-24h) only docked mucocysts seem to accumulate in the cell . However, by 36h a considerable increase seems to have taken place, particularly in the number of mucocysts located in the cytoplasm . In the cycloheximide-treated cells this increase in mucocysts begins to be blocked after 6h of exposure to the drug . These observations are in agreement with the results obtained from the freeze-fracture data on the concomitant increase in number of rosettes . This system therefore offers the first possibility of exploring the biosynthesis of these components. Appl Environ Microbiol, 1985 Oct, 50(4), 906 - 13 Identification of thermophilic bacteria in solid-waste composting; Strom PF; The thermophilic microbiota of solid-waste composting, with major emphasis on Bacillus spp., was examined with Trypticase soy broth (BBL Microbiology Systems) with 2% agar as the initial plating medium . Five 4.5-liter laboratory units at 49 to 69 degrees C were fed a mixture of dried table scraps and shredded newspaper . The composting plants treating refuse at Altoona, Pa., and refuse-sludge at Leicester, England, were also sampled . Of 652 randomly picked colonies, 87% were identified as Bacillus spp . Other isolates included two genera of unidentified nonsporeforming bacteria (one of gram-negative small rods and the other of gram-variable coccobacilli), the actinomycetes Streptomyces spp . and Thermoactinomyces sp., and the fungus Aspergillus fumigatus . Among the Bacillus isolates, the following, in order of decreasing frequency, were observed: B . circulans complex, B . stearothermophilus, B . coagulans types A and B, B . licheniformis, B . brevis, B . sphaericus, Bacillus spp . types i and ii, and B . subtilis . About 15% of the Bacillus isolates could be assigned to species only by allowing for greater variability in one or more characteristics than has been reported by other authors for their strains . In particular, growth at higher temperatures than previously reported was found for strains of several species . A small number of Bacillus isolates (less than 2%) could not be assigned to any recognized species. Appl Environ Microbiol, 1985 Oct, 50(4), 899 - 905 Effect of temperature on bacterial species diversity in thermophilic solid-waste composting; Strom PF; Continuously thermophilic composting was examined with a 4.5-liter reactor placed in an incubator maintained at representative temperatures . Feed was a mixture of dried table scraps and shredded newspaper wetted to 55% moisture . One run at 49 degrees C (run A) employed a 1:4 feed-to-compost ratio, while the other runs used a 10:1 ratio and were incubated at 50, 55, 60, or 65 degrees C . Due to self-heating, internal temperatures of the composting mass were 0 to 7 degrees C hotter than the incubator . Two full-scale composting plants (at Altoona, Pa., and Leicester, England) were also examined . Plate counts per gram (dry weight) on Trypticase soy broth (BBL Microbiology Systems) with 2% agar ranged from 0.7 X 10(9) to 5.3 X 10(9) for laboratory composting and 0.02 X 10(9) to 7.4 X 10(9) for field composting . Fifteen taxa were isolated, including 10 of genus Bacillus, which dominated all samples except that from run A . Species diversity decreased markedly in laboratory composting at 60 degrees C and above, but was similar for the three runs incubated at 49, 50, and 55 degrees C . The maximum desirable composting temperature based on species diversity is thus 60 degrees C, the same as that previously recommended based on measures of the rate of decomposition. Appl Environ Microbiol, 1985 Oct, 50(4), 777 - 80 Galactokinase activity in Streptococcus thermophilus; Hutkins R et al.; ATP-dependent phosphorylation of {14C}galactose by 11 strains of Streptococcus thermophilus indicated that these organisms possessed the Leloir enzyme, galactokinase (galK) . Activities were 10 times higher in fully induced, galactose-fermenting (Gal+) strains than in galactose-nonfermenting (Gal-) strains . Lactose-grown, Gal- cells released free galactose into the medium and were unable to utilize residual galactose or to induce galK above basal levels . Gal+ S . thermophilus 19258 also released galactose into the medium, but when lactose was depleted growth on galactose commenced, and galK increased from 0.025 to 0.22 micromol of galactose phosphorylated per min per mg of protein . When lactose was added to galactose-grown cells of S . thermophilus 19258, galK activity rapidly decreased . These results suggest that galK in Gal+ S . thermophilus is subject to an induction-repression mechanism, but that galK cannot be induced in Gal- strains. Appl Environ Microbiol, 1985 Oct, 50(4), 772 - 6 Galactose transport in Streptococcus thermophilus; Hutkins R et al.; Although Streptococcus thermophilus accumulated {14C}lactose in the absence of an endogenous energy source, galactose-fermenting (Gal+) cells were unable to accumulate {14C}galactose unless an additional energy source was added to the test system . Both Gal+ and galactose-nonfermenting (Gal-) strains transported galactose when preincubated with sucrose . Accumulation was inhibited 50 or 95% when 10 mM sodium fluoride or 1.0 mM iodoacetic acid, respectively, was added to sucrose-treated cells, indicating that ATP was required for galactose transport activity . Proton-conducting ionophores also inhibited galactose uptake, although N,N'-dicyclohexyl carbodiimide had no effect . The results suggest that galactose transport in S . thermophilus occurs via an ATP-dependent galactose permease and that a proton motive force is involved . The galactose permease in S . thermophilus TS2b (Gal+) had a Km for galactose of 0.25 mM and a Vmax of 195 micromol of galactose accumulated per min per g (dry weight) of cells . Several structurally similar sugars inhibited galactose uptake, indicating that the galactose permease had high affinities for these sugars. Tohoku J Exp Med, 1985 Oct, 147(2), 135 - 44 Determination by enzyme-linked immunosorbent assay (ELISA) of specific IgG antibody activities for diagnosis of farmer's lung disease; Konishi K et al.; The specific IgG antibodies to thermophilic actinomycetes in the sera from patients with farmer's lung disease were quantitated by an ELISA and the results thereof were compared with those obtained by the standard double immunodiffusion assay (Ouchterlony's method) . All sera from patients with farmer's lung disease were precipitin positive to Thermoactinomyces vulgaris and/or Micropolyspora faeni by the Ouchterlony's method, and revealed significantly high levels of IgG antibody activities against these antigens by ELISA . We have found 18 precipitin-positive but asymptomatic subjects in a survey of the dairy farming population of the northern area of IWATE Prefecture . Their sera were precipitin positive to antigens related to the farmer's lung disease, however IgG antibody activities as determined by ELISA to these antigens was significantly lower in the group of asymptomatics than in the group of symptomatics . Precipitin negative dairy farming controls and normal controls living in the urban area revealed low antibody activities as determined by ELISA . From these studies, we concluded that the assay of specific IgG antibody activity to thermophilic actinomyces by ELISA is useful for the diagnosis and screening of farmer's lung disease. Genetics, 1985 Oct, 111(2), 273 - 86 Isolation and genetic analysis of mutations at the SerH immobilization antigen locus of Tetrahymena thermophila; Doerder FP et al.; Multiple alleles at the SerH locus specify the major cell surface protein (immobilization antigen) of the ciliate Tetrahymena thermophila . Following mutagenesis of SerH1 homozygotes, two mutations, H1-1 and H1-2, were recovered in heterozygous form . Mutant homozygotes do not express H1 antigen, nor is H1 expressed in F1 progeny of crosses to wild-type strains homozygous for SerH2 or SerH3 . H1-1 and H1-2 segregate without recombination from these wild-type alleles in expected F2 and testcross Mendelian ratios . H1-1 and H1-2 do, however, complement each other to express H1 antigen . Experiments suggest this complementation is due neither to recombination during macronuclear development nor to interallelic complementation of defective SerH1 gene products . These results suggest that SerH1 is intact in one mutant, and possibly both, although no such allele has been segregated in testcross progeny (N = 205) . The hypothesis is presented that complementation between H1-1 and H1-2 is due to interaction between allele-specific regulators closely linked to the SerH1 gene. J Bacteriol, 1985 Oct, 164(1), 421 - 4 Uracil-DNA glycosylase of thermophilic Thermothrix thiopara; Kaboev OK et al.; An activity which released free uracil from dUMP-containing DNA was purified approximately 1,700-fold from extracts of Thermothrix thiopara, the first such activity to be isolated from extremely thermophilic bacteria . The enzyme appeared homogeneous, according to the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis . It had a native molecular weight of 26,000 and existed as a monomer protein in water solution . The enzyme had an optimal activity at 70 degrees C, between pH 7.5 and 9.0, and in the presence of 0.2% Triton X-100 . It had no cofactor requirement and was not inhibited by EDTA, but it was sensitive to N-ethylmaleimide . The purified enzyme did not contain any nuclease that acted on native or depurinated DNA . The Arrhenius activation energy was 76 kJ/mol between 30 and 50 degrees C and 11 kJ/mol between 50 and 70 degrees C . The rate of heat inactivation of the enzyme followed first-order kinetics with a half-life of 2 min at 70 degrees C . Ammonium sulfate and bovine serum albumin protected the enzyme from heat inactivation . One T . thiopara cell contains enough activity to release about 2 X 10(8) uracil residues from DNA during one generation time at 70 degrees C. J Gen Microbiol, 1985 Oct, 131 ( Pt 10), 2789 - 94 Isolation of thermophilic mutants of Bacillus subtilis and Bacillus pumilus and transformation of the thermophilic trait to mesophilic strains; Droffner ML et al.; Thermophilic mutants were isolated from mesophilic Bacillus subtilis and Bacillus pumilus by plating large numbers of cells and incubating them for several days at a temperature about 10 degrees C above the upper growth temperature limit for the parent mesophiles . Under these conditions we found thermophilic mutant strains that were able to grow at temperatures between 50 degrees C and 70 degrees C at a frequency of less than 10(-10) . The persistence of auxotrophic and antibiotic resistance markers in the thermophilic mutants confirmed their mesophilic origin . Transformation of genetic markers between thermophilic mutants and mesophilic parents was demonstrated at frequencies of 10(-3) to 10(-2) for single markers and about 10(-7) for two unlinked markers . With the same procedure we were able to transfer the thermophilic trait from the mutant strains of Bacillus to the mesophilic parental strains at a frequency of about 10(-7), suggesting that the thermophilic trait is a phenotypic consequence of mutations in two unlinked genes. J Cell Sci, 1985 Oct, 78, 49 - 65 Protein secretion in Tetrahymena thermophila: characterization of the secretory mutant strain SB281; Maihle NJ et al.; The ciliated protozoon Tetrahymena thermophila contains membrane-bounded secretory organelles termed mucocysts, the release of which has previously been characterized ultrastructurally as a model system for the events occurring during membrane fusion and protein secretion . Recently, a series of secretory mutant strains of Tetrahymena has been isolated following mutagenesis of a parental wild-type strain designated SB210 . In this study, the correlates of non-release in one unique mutant strain of this series, designated SB281, are described . SB281 appears to express a diminished (undetectable) level of the major 34000 Mr proteinaceous secretory product of Tetrahymena, as determined by Western immunoblot analysis and indirect immunofluorescence labelling . Thin-section electron-microscopic studies of these cells reveal that they possess no docked or free mature mucocysts . In addition, freeze-fracture electron microscopy demonstrates that an intramembrane particle array termed the rosette, present in the plasma membrane of wild-type cells above sites of docked mucocysts, is absent in the plasma membrane of mutant SB281 cells . A morphometric analysis of intramembrane particles in the plasma membrane of both wild-type and mutant cells indicates that both strains have a similar intramembrane particle density in both leaflets of the the plasma membrane . Although assembled rosettes are missing in the plasma membrane of mutant cells, a 15 nm intramembrane particle size class does exist in the plasma membrane of the mutant, but this size class is significantly reduced in number relative to wild-type. Proc Natl Acad Sci U S A, 1985 Oct, 82(20), 6908 - 12 Multiple forms of tubulin in the cilia and cytoplasm of Tetrahymena thermophila; Suprenant KA et al.; Most higher eukaryotic tubulins are separated into alpha- and beta-tubulin when electrophoresed in NaDodSO4- denaturing gels, while many lower eukaryotic tubulins are poorly resolved under these conditions, which include a stacking gel (pH 6.80) and a separating gel (pH 8.80) . By lowering the pH of the separating gel to 8.25, we have found that tubulin isolated from the protozoan Tetrahymena thermophila is resolved by one-dimensional polyacrylamide gel electrophoresis into two alpha-tubulins and one beta-tubulin . Moreover, at least five alpha- and two beta-tubulin isotypes are identified in Tetrahymena by isoelectric focusing and two-dimensional polyacrylamide gel electrophoresis . Three of these alpha isotypes and one beta isotype are found specifically in ciliary microtubules, while the other two isotypes are found only in the cytoplasmic tubulin pool that was isolated and induced to self-assemble into microtubules in vitro . Peptide mapping by limited proteolytic digestion indicates that the tubulins are closely related . Possible mechanisms for the generation and selection of these tubulin isotypes are discussed. Appl Environ Microbiol, 1985 Oct, 50(4), 1103 - 6 Isolation and partial characterization of plasmid DNA from Streptococcus thermophilus; Herman RE et al.; Of 23 strains of Streptococcus thermophilus examined, 5 were found to contain a single small cryptic plasmid, designated pHM1 through pHM5 . Through analysis by restriction endonuclease mapping and DNA-DNA hybridization, the five plasmids were found to be closely related . They were present in 4 to 18 copies per cell and ranged in size from 1.4 to 2.2 megadaltons . Plasmids pHM1 and pHM5, as well as pHM2 and pHM4, were found to be identical . Single restriction endonuclease sites were observed on each plasmid for PvuII and MboI . Plasmids pHM2 and pHM3 each had an additional single site for HhaI, and pHM1 had an additional single site for HindIII . The characteristics of these plasmids may make them useful for the development of cloning vectors for use in S . thermophilus. Eur J Cell Biol, 1985 Sep, 38(2), 306 - 11 Rapid and sensitive assays for phagosomal acidification in Paramecium and Tetrahymena; Fok AK et al.; Biochemical and cytochemical procedures were developed to measure the rate of phagosomal acidification for phagosomal pH ranging from 5 to 2.5 . These assays were based on the pH-dependent inactivation with time of horseradish peroxidase (HRP) activity, a result attributable to the dissociation of this enzyme to a colorless protein and ferriprotoporphyrin in acidic solutions . When preincubated in buffers of varying pH, the rate of HRP inactivation followed a sigmoid curve, with the highest rate of inactivation between 4.3 and 3.5 when using citrate-phosphate buffer and between pH 3.4 and 2.8 when using the universal ABC buffer . This inactivation was temperature but not concentration dependent . When Paramecium caudatum, members of the P . aurelia complex or Tetrahymena thermophila was pulsed briefly with HRP and small fluorescent beads, the loss of HRP activity, measured biochemically in cell homogenates and/or cytochemically in phagosomes, was rapid and followed the kinetics of a first-order rate reaction . Both assays gave similar values for the rate constant for acidification and similar rates of inhibition when P . caudatum was exposed to a proton ionophore, carbonyl cyanide p-trifluoromethoxyphenyl hydrazone . These assays can readily be adapted to other phagocytic cells as long as a rapid procedure is available for removing all unphagocytosed HRP and latex beads . These procedures are sensitive and rapid thus allowing many samples to be quickly prepared and analyzed. J Bacteriol, 1985 Sep, 163(3), 1275 - 8 Thermoanaerobacter ethanolicus in a comparison of the growth efficiencies of thermophilic and mesophilic anaerobes; Lacis LS et al.; Maintenance coefficients and theoretical maximum growth yields, with respect to both substrate and ATP, were estimated for Thermoanaerobacter ethanolicus growing in a glucose-limited, continuous culture . A comparison of these values with those for other bacteria showed that, contrary to predictions by others, anaerobic thermophiles had neither low observed growth yields nor high maintenance energy coefficients. Ital J Biochem, 1985 Sep-Oct, 34(5), 341 - 55 Comparative study of glucosidases from the thermophilic fungus Thermoascus aurantiacus Miehe . Purification and characterization of intracellular beta-glucosidase; Bedino S et al.; Intracellular beta-glucosidase was extracted from the mycelium of Th . aurantiacus, concentrated by DEAE-cellulose treatment, separated from alpha-glucosidase by hydroxylapatite chromatography and purified to electrophoretic homogeneity . Optimally active at 75 degrees C and pH 4.2, beta-glucosidase displayed complex kinetics with p-nitrophenyl-beta-glucoside which inhibited the enzyme at concentrations greater than 0.5 mM . With cellobiose the kinetics were practically hyperbolic at 70 degrees C (Hill coefficient nH = 1.09 and Km = 0.83 mM), but faint inhibition was observed at 50 degrees C . beta-glucosidase shares with alpha-glucosidase a high number of physicochemical properties: with similar aminoacid composition, very close isoelectric point (4.5 and 4.2), high molecular weight in the native state (175,000 and 140,000), the two enzymes showed the same behaviour on DEAE-cellulose, were equally stable at high temperature and were dissociated by 6 M urea to still active proteins . Furthermore, the carbohydrate contents of beta-glucosidase (17.6%) is not far from that previously determined for some forms of alpha-glucosidase (14-16%). Arch Biochem Biophys, 1985 Sep, 241(2), 571 - 6 A specific L-asparaginase from Thermus aquaticus; Curran MP et al.; The L-asparaginase from an extreme thermophile, Thermus aquaticus strain T351, was highly substrate- and stereospecific, with no activity against glutamine or D-asparagine . It had a high Km of 8.6 mM . In these aspects it closely resembled the corresponding enzymes from thermophilic bacteria . The enzyme had a molecular weight of 80,000, an isoelectric point of 4.6, and a pH optimum of 9.5 . It showed some substrate inhibition above 20 mM asparagine and was also inhibited by L-aspartic acid, D- and L-lysine (Ki of 5.2 and 1.25 mM, respectively), and D- and L-serine . The half-life of the enzyme at 85 degrees C was 40 min . The Arrhenius plot showed a change in slope at 55 degrees C. Nucleic Acids Res, 1985 Aug 26, 13(16), 5817 - 31 Macronuclear DNA of Tetrahymena thermophila exists as defined subchromosomal-sized molecules; Altschuler MI et al.; Using the method of orthogonal-field-alternation gel electrophoresis, we have resolved the macronuclear DNA of Tetrahymena thermophila into a series of distinct bands . Using electrode switching intervals ranging from 10 to 70 seconds we have resolved DNA bands ranging in size from about 21 kb up to and beyond the size of yeast chromosomes VII and XV . Hybridization of Southern blots from these gels to both unique and repetitive DNA sequences shows that the macronuclear genome of T . thermophila has a precise organization . The unique sequences tested each hybridize to only one band of macronuclear DNA and the hybridization patterns seem to be identical in several inbred strains examined. J Biol Chem, 1985 Aug 25, 260(18), 10013 - 8 Regulation of galactokinase gene expression in Tetrahymena thermophila . II . Identification of 3,4-dihydroxyphenylalanine as a primary effector of adrenergic control of galactokinase expression; Ness JC et al.; Intracellular concentrations of catecholamines were determined in wild-type and mutant Tetrahymena thermophila, using the highly sensitive techniques of high-performance liquid chromatography and electro-chemical detection . Catecholamines were determined in these cell strains grown under various steady-state conditions, including those which initiate and maintain repression of galactokinase gene expression . Wild-type cells grown in defined minimal medium supplemented with 1% glycerol, exhibiting derepressed galactokinase synthesis, were found to contain considerable quantities of dopa (3,4-dihydroxyphenylalanine) and dopamine, but no detectable levels of either norepinephrine or epinephrine . Analyses of wild-type cells revealed a strong positive correlation between the internal concentration of dopa and expression of the galactokinase gene, both of which are regulated by exogenous carbohydrates, catecholamine agonists, or dibutyryl-cAMP; an analogous relationship between intracellular dopamine concentrations and galactokinase activity was not found . In addition, a correlation between intracellular dopa content and the phenotypic expression of galactokinase in various mutants deficient in the catecholamine biosynthetic pathway or in glucokinase further confirms the role of dopa as a primary effector in the regulation of galactokinase gene expression. J Biol Chem, 1985 Aug 25, 260(18), 10001 - 12 Regulation of galactokinase gene expression in Tetrahymena thermophila . I . Intracellular catecholamine control of galactokinase expression; Ness JC et al.; The addition of glucose to the medium of Tetrahymena thermophila results in a 7-fold repression of galactokinase (EC 2.7.1.6; ATP:D-galactose-1-phosphotransferase) . The presence of millimolar amounts of the catecholamines dopa, dopamine, norepinephrine, and epinephrine or the hormone glucagon also results in the repression of galactokinase in the absence of glucose . The addition of millimolar amounts of adrenergic agonists (isoproterenol, tyramine, 2-amino-6,7-dihydroxytetrahydronaphthalene) results in significant repression of galactokinase in the absence of glucose; concentrations of 2-amino-6,7-dihydroxytetrahydronaphthalene less than or equal to 10(-4) M result in a derepression of galactokinase specific activity . Addition of adrenergic antagonists (propranolol, dichloroisoproterenol) have no effect on galactokinase activity at concentrations less than 10(-4) M but do arrest cell growth at greater concentrations . The addition of the cAMP analogs caffeine or theophylline in millimolar amounts results in repression of galactokinase activity; however, cell growth is greatly slowed or completely arrested at these concentrations . Analysis of the repression response of several mutants demonstrates that mutants deficient in catecholamine biosynthesis are altered in their regulation of galactokinase . Measurements of intracellular cAMP levels for 0-24 h following the addition of several of the above compounds to exponentially growing cells did not demonstrate any change over this period . Measurement of intracellular cAMP levels for 24 h following the addition of glucose or galactose to exponentially growing wild-type and mutant cell strains did not demonstrate any difference in cAMP concentrations over this period although a wide range of galactokinase activity was exhibited . Starvation of wild-type cells prior to the addition of glucose in minimal medium without added carbohydrate resulted in a significant increase in cAMP following the addition of glucose . This increase is demonstrated to be dependent upon the ability of the cells to resume division after the arrest of growth and is not correlated with galactokinase regulation . These results support the conclusion that cAMP is not involved in the repression of galactokinase gene expression initiated by glucose or hormone-like effectors and demonstrate the participation of an adrenergic control system in galactokinase regulation which is subordinate to the regulation by glucose . A possible model is discussed. J Protozool, 1985 Aug, 32(3), 517 - 9 An organofluorophosphate-hydrolyzing activity in Tetrahymena thermophila; Landis WG et al.; An enzymatic activity that hydrolyzes O,O-diisoproplyphosphofluoridate (DFP) and O-1,2,2-trimethylpropylmethylphosphonofluoridate (Soman) was discovered in the ciliate protozoan Tetrahymena thermophila . The enzymatic activity classifies the protein as Mazur-type similar to that found in hog kidney and Escherichia coli . The rate of hydrolysis of Soman by the Tetrahymena-extract is the highest, on a per gram of extract basis, of any eucaryote . The molecular weight is approximately 75,400 as determined by Sephacryl column chromatography . A maximum fifteen-fold purification has been achieved . Potential exists for the detoxification and one-step detection of common organofluorophosphate pollutants . Additionally, Tetrahymena should prove an easier subject for manipulation than mammalian or squid sources . Protozoa may be a potentially important source of detoxification and degradation enzymes for other environmental contaminants. J Dairy Sci, 1985 Aug, 68(8), 1910 - 6 Use of microbial beta-lactamase to destroy penicillin added to milk; Korycka-Dahl M et al.; A simple method is described for destruction of penicillin residues in bulk milk to an undetectable amount (less than .003 U/ml) with commercially available crude beta-lactamase enzyme . Milk containing .1 or .5 U/ml penicillin G was treated with .01 to 1.0 mU/ml of beta-lactamase (Bacillus cereus) for up to 96 h . The Bacillus stearothermophilus var . calidolactis assay was used to quantify penicillin in milk between .003 to 1.0 U/ml . The .5 U/ml of penicillin G was reduced to an undetectable amount within 18 h at 4 degrees C by 1.0 mU/ml of beta-lactamase . The development of titratable acidity over 5 to 6 h in contaminated milks treated with beta-lactamase and inoculated with Streptococcus thermophilus GH, Streptococcus cremoris, Streptococcus lactis, or a commercial starter culture was the same as for control milk samples containing no additives or only enzyme . Pilot-scale manufacture of Swiss and Cheddar cheeses from contaminated milks treated with beta-lactamase yielded cheeses of comparable quality, to control cheeses produced from penicillin-free milk . There were no delays in acid production as judged from pH measurements during production and ripening of the cheeses . About 50% of beta-lactamase activity added to milk remained after pasteurization at 63 degrees C for 30 min . The safety for human consumption of cheese containing small quantities of penicillin degradation products from milk treated with beta-lactamase remains to be established. Mol Cell Biol, 1985 Aug, 5(8), 1925 - 32 Expression of a cell surface immobilization antigen during serotype transformation in Tetrahymena thermophila; Williams NE et al.; A temperature shift from 40 to 28 degrees C rapidly induced expression of a specific immobilization antigen at the cell surface in Tetrahymena thermophila . This transformation was inhibited by actinomycin D and cycloheximide but not by colchicine or cytochalasin B . The major surface antigen expressed at 28 degrees C in cells homozygous for the SerH3 allele was partially purified, and an antiserum against this preparation was raised in rabbits . Electrophoresis, immunoblot, and {35S}methionine incorporation studies are reported which support the conclusion that the H3 antigen is an acidic protein with an Mr of approximately 52,000 daltons . An induced synthesis of the H3 immobilization antigen was detected within 30 min after a shift from 40 to 28 degrees C . This protein appeared to be synthesized in the microsomal fraction and transferred without cleavage to the cell surface, where it was inserted first into nonciliated regions. Mol Cell Biol, 1985 Aug, 5(8), 2061 - 9 Induction of acquired thermotolerance in Tetrahymena thermophila: effects of protein synthesis inhibitors; Hallberg RL et al.; When Tetrahymena thermophila cells growing at 30 degrees C are shifted to either 40 or 43 degrees C, the kinetics and extent of induction of heat shock mRNAs in both cases are virtually indistinguishable . However, the cells shifted to 40 degrees C show a typical induction of heat shock protein (HSP) synthesis and survive indefinitely (100% after 24 h), whereas those at 43 degrees C show an abortive synthesis of HSPs and die (less than 0.01% survivors) within 1 h . Cells treated at 30 degrees C with the drugs cycloheximide or emetine, at concentrations which are initially inhibitory to protein synthesis and cell growth but from which cells can eventually recover and resume growth, are after this recovery able to survive a direct shift from 30 to 43 degrees C (ca . 70% survival after 1 h) . This induction of thermotolerance by these drugs is as efficient in providing thermoprotection to cells as is a prior sublethal heat treatment which elicits the synthesis of HSPs . However, during the period when drug-treated cells recover their protein synthesis ability and simultaneously acquire the ability to subsequently survive a shift to 43 degrees C, none of the major HSPs are synthesized . The ability to survive a 1-h, 43 degrees C heat treatment, therefore, does not absolutely require the prior synthesis of HSPs . But, as extended survival at 43 degrees Celsius depends absolutely on the ability of cells to continually synthesize HSPs, it appears that a prior heat shock as well as the recovery from protein synthesis inhibition elicits a change in the protein synthetic machinery which allows the translation of HSP mRNAs at what would otherwise be a nonpermissive temperature for protein synthesis. Mol Cell Biol, 1985 Aug, 5(8), 2039 - 50 Reproducible and variable genomic rearrangements occur in the developing somatic nucleus of the ciliate Tetrahymena thermophila; Howard EA et al.; We analyzed the extent, reproducibility, and developmental control of genomic rearrangements in the somatic macronucleus of the ciliate Tetrahymena thermophila . To exclude differences caused by genetic polymorphisms, we constructed whole-genome homozygotes, and we compared the homozygous progeny derived from single macronuclear differentiation events . This strategy enabled us to identify a novel form of variable rearrangement and to confirm previous findings that rearranged sequences occur at a high frequency in the Tetrahymena genome . Rearrangements studied here were deletions of both unique and interchromosomally dispersed repetitive DNA sequences involving DNA rejoining of internal, nontelomeric regions of macronuclear DNAs . We showed that although rearrangements of some sequence classes are reproducible among independently developed macronuclei, other specific sequence classes are variably rearranged in macronuclear development . The variable somatic genomes so produced may be the source of phenotypically variant cell lines. J Protozool, 1985 Aug, 32(3), 480 - 5 Updating rDNA restriction enzyme maps of Tetrahymena reveals four new intron-containing species; Nielsen H et al.; The extrachromosomal rDNA molecules from a number of Tetrahymena strains were characterized by restriction enzyme mapping using three different restriction enzymes combined with gel blotting and hybridization analysis . Strains from four out of six recently described species were found to contain an intron in the 26s rRNA coding region . The evolutionary relationship among the species of the T . pyriformis complex was examined on the basis of the rDNA maps with emphasis on similarities between two of the new species and the widely studied T . thermophila and T . pigmentosa . Examination of a large number of T . pigmentosa strains showed this species to exhibit an unusual polymorphism with respect to its rDNA . It is suggested that recombinational cross-over events play a role in the formation of new rDNA alleles in this species. J Mol Biol, 1985 Jul 20, 184(2), 257 - 77 X-ray crystallographic structure of the light-harvesting biliprotein C-phycocyanin from the thermophilic cyanobacterium Mastigocladus laminosus and its resemblance to globin structures; Schirmer T et al.; The structure of the biliprotein C-phycocyanin from the thermophilic cyanobacterium Mastigocladus laminosus has been determined at 3 A resolution by X-ray diffraction methods . Phases have been obtained by the multiple isomorphous replacement method . The electron density map could be improved by solvent flattening and has been interpreted in terms of the amino acid sequence . The protein consists of three identical (alpha-beta)-units which are arranged around a threefold symmetry axis to form a disc of approximate dimensions 110 A X 30 A with a central channel of 35 A in diameter . This aggregation form is supposed to be the same as that found in the rods of native phycobilisomes . Both subunits, alpha and beta, exhibit a similar structure and are related by a local twofold rotational axis . Each subunit is folded into eight helices and irregular loops . Six helices are arranged to form a globular part, whereas two helices stick out and mediate extensive contact between the subunits . The arrangement of the helices of the globular part resembles the globin fold: 59 equivalent C alpha-atoms have a root-mean-square deviation of 2 X 9 A . The chromophores attached to cystein 84 of the alpha- and beta-subunits are topologically equivalent to the haem . All three chromophores of C-phycocyanin, open-chain tetrapyrroles, are in an extended conformation . alpha 84 and beta 84 are attached to helix E (globin nomenclature), beta 155 is linked to the G--H loop . The shortest centre-to-centre distance between chromophores in trimer is 22 A. Acta Odontol Scand, 1985 Jul, 43(3), 147 - 53 Secretory and serum antibodies against Streptococcus lactis, Streptococcus thermophilus, and Lactobacillus bulgaricus in relation to ingestion of fermented milk products; Carlsson P et al.; Serum, saliva, and urine were analyzed for the presence of IgA, IgG, and IgM antibodies reactive with the yoghurt bacteria Streptococcus thermophilus and Lactobacillus bulgaricus . A comparison was made between four subjects who frequently ate yoghurt and four subjects who never ate yoghurt . Salivary IgA and serum IgG activity against the milk-fermenting bacterium S . lactis was studied in five other subjects before, during, and after a period of ingestion of a fermented milk product, filmjolk . All analyses were carried out by an enzyme-linked immunosorbent assay method . Antibody activity against the yoghurt bacteria was found in saliva, serum, and urine . No difference in antibody activity between yoghurt eaters and non-yoghurt eaters was measured for salivary IgA, but for serum IgG a lower activity against S . thermophilus was present among the yoghurt eaters . Antibody activity against S . lactis was present already before the ingestion of filmjolk began, and the activity was not altered during the period of ingestion . It is concluded that in adult subjects, the ingestion of milk-fermenting bacteria does not result in a significant change in the antibody activity against these bacteria. Biochimie, 1985 Jul-Aug, 67(7-8), 841 - 7 A modified two primer approach to oligonucleotide-directed in vitro mutagenesis; Sims PF et al.; Using oligonucleotide-directed mutagenesis, we are trying to define the features of the protein structure that are important for the DNA and c-AMP binding by CAP from E . coli, the enzymic activity and putative DNA binding of dihydrofolate reductase of L . casei, and the functionally important regions of the self-splicing RNA of the r-RNA intron of Tetrahymena thermophila . We have used a modification of the method described by Norris et al . {1} . A mutagenic primer and an M13 universal sequencing primer are annealed simultaneously to a template from an M13 clone containing the DNA to be mutagenised and, after DNA strand extension, the fragment is cut out and recloned into either M13 or plasmid vectors . We have analysed the effect on the frequency of mutation of: the temperature used for strand extension; the class of base change attempted; the host mismatch repair system . A recently developed system for phenotypic detection of mutations in the Tetrahymena intron aided in determining mutation frequencies. Vet Res Commun, 1985 Jul, 9(3), 167 - 98 The occurrence and significance of Campylobacter jejuni in man and animals; Shane SM et al.; Campylobacter jejuni, which is now recognized as a discrete species, is a gram negative, microaerophilic, thermophilic, nalidixic acid sensitive, hippurate positive pathogen requiring special selective media for propogation . The organism is widely distributed in avian species, experimental and companion animals and in humans . Mammalian campylobacteriosis is characterized by an enterocolitis of variable severity . The prevalence of the condition is relatively high in young individuals, in underdeveloped countries and in subjects with diarrhea . Food animals, especially poultry, are reservoirs of the organism and infection occurs following consumption of untreated surface water, unpasteurized milk, incompletely cooked meat or other contaminated food products . Close contact with infected immature companion animals is a significant cause of campylobacteriosis in children and direct intrafamilial transmission and occupational infection have been documented . Campylobacteriosis attributable to C . jejuni is a condition of emerging significance which arises principally from deficiencies in hygiene inherent in the environment and in the food chain which extends from domestic animals to the consumer. J Biochem (Tokyo), 1985 Jul, 98(1), 95 - 103 Complete nucleotide sequence of a thermophilic alpha-amylase gene: homology between prokaryotic and eukaryotic alpha-amylases at the active sites; Ihara H et al.; The nucleotide sequence of a thermophilic, liquefying alpha-amylase gene cloned from B . stearothermophilus was determined . The NH2-terminal amino acid sequence analysis of the B . stearothermophilus alpha-amylase confirmed that the reading frame of the gene consisted of 1,644 base pairs (548 amino acids) . The B . stearothermophilus alpha-amylase had a signal sequence of 34 amino acids, which was cleaved at exactly the same site in E . coli . The mature enzyme contained two cysteine residues, which might play an important role in maintenance of a stable protein conformation . Comparison of the amino acid sequence inferred from the B . stearothermophilus alpha-amylase gene with those inferred from other bacterial liquefying alpha-amylase genes and with the amino acid sequences of eukaryotic alpha-amylases showed three homologous sequences in the enzymatically functional regions. Can J Microbiol, 1985 Jul, 31(7), 614 - 9 Genetic relationship between pUB110 and antibiotic-resistant plasmids obtained from thermophilic bacilli; Hoshino T et al.; The molecular relationship between pUB110 (Kmr, 4.4 kilobases (kb} and antibiotic-resistant plasmids from thermophilic bacilli, pTHT15 (Tcr, 4.5 kb) and pTHN1 (Kmr, 4.8 kb), were studied by blot hybridization . Extensive homology was observed between pUB110 and pTHT15 at the region which includes the replication origin . Incompatibility studies revealed that pTHT15 and pUB110 were slightly incompatible in Bacillus subtilis but that they were apparently compatible in B . stearothermophilus . This difference in incompatibility between pTHT15 and pUB110 in the two host cells might be due to a difference in the copy number of pTHT15 in the two organisms . From the results of blot hybridization, mode of kanamycin inactivation, and DNA sequencing, it was determined that pTHN1 encoded the identical gene for kanamycin nucleotidyl transferase as that of pUB110 . All three plasmids pTHT15, pTHN1, and pUB110 shared a common DNA homology at the in vitro membrane-binding region. Proc Natl Acad Sci U S A, 1985 Jul, 82(14), 4758 - 62 Tetrahymena thermophila glutamine tRNA and its gene that corresponds to UAA termination codon; Kuchino Y et al.; Nucleotide sequence analysis of one of several tRNA genes cloned from Tetrahymena thermophila macronuclear DNA indicated that it corresponds to a tRNA species having TTA as anticodon . Subsequently, the tRNA species corresponding to that gene was isolated and its nucleotide sequence was determined by post-labeling techniques . The nucleotide sequence was found to be pG-G-U-U-C-C-A-U-A-m2G-U-A-psi-A-G-D-G-G-D- D-A-G-U-A-C-U-G-G-G-G-A-Cm-U-Um-U-A-i6A-A-psi-C-C-C-U-U-G-A-C- m5C-U-G-G-G-U-psi-C-G-m1A-A-U-C-C-C-A-G-U-G-G-G-A-C-C-U-C-C-AOH . This tRNA sequence exactly matched the DNA sequence of the corresponding tRNA gene . The first position of anticodon is 2'-O-methyluridine (Um), forming UmUA as the anticodon, which presumably recognizes the ochre termination codon UAA . This tRNA species is aminoacylated with glutamine by a Tetrahymena crude aminoacyl tRNA synthetase fraction, suggesting that ochre termination codon is used as a glutamine codon during cytoplasmic protein synthesis in Tetrahymena. Eur J Biochem, 1985 Jul 1, 150(1), 61 - 6 Nucleotide sequence of the Escherichia coli gap gene . Different evolutionary behavior of the NAD+-binding domain and of the catalytic domain of D-glyceraldehyde-3-phosphate dehydrogenase; Branlant G et al.; A 1523-base-pair DNA fragment, spanning the gap gene from Escherichia coli, has been sequenced . It contains an open-reading frame whose length (330 amino acids) is in agreement with D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) molecular mass . This coding sequence is preceded by a Shine-Dalgarno complementary sequence and by two overlapping promoter-like structures . The codon usage within gap is consistent with that expected for a gene which is strongly expressed . The amino acid sequence of the E . coli GAPDH, deduced from the DNA sequence, contains all the amino acids postulated to play a functional role in GAPDH . Comparison of the E . coli enzyme with enzymes from other species reveals different evolutionary behaviour of the NAD+-binding domain and of the catalytic domain of GAPDH . The E . coli enzyme is found to be more similar to eucaryotic enzymes than to enzymes from thermophilic bacteria . This observation is discussed in terms of adaptation to growth at high temperature. J Appl Biochem, 1985 Jun, 7(3), 192 - 201 Purification and properties of glucose-6-phosphate dehydrogenase from Bacillus stearothermophilus; Okuno H et al.; Glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ 1-oxidoreductase, EC 1.1.1.49) from the thermophilic bacteria, Bacillus stearothermophilus, was purified and its properties were examined . The enzyme was shown to consist of four identical subunits, each of about Mr 50,000 . This enzyme utilized both NADP+ and NAD+ as cofactors, and the maximum velocity for both cofactors was similar . However, the Km values were quite different from each other, being 0.016 and 1.64 mM for NADP+ and NAD+, respectively . From the analysis of sulfhydryl groups it was shown that there is one sulfhydryl group and one disulfide bridge per subunit . This sulfhydryl group had no reactivity with 5,5'-dithiobis(2-nitrobenzoic acid) in the absence of guanidine hydrochloride . The enzyme showed a remarkable thermostability as well as storage stability. Biochem Int, 1985 Jun, 10(6), 955 - 62 Thermophilic NAD-dependent glutamate dehydrogenase from Bacillus stearothermophilus; Mantsala P; A rapid purification procedure for glutamate dehydrogenase (GDH) from Bacillus stearothermophilus var calidolactis was developed . The homogeneous enzyme with a total molecular weight of approximately 240,000 daltons, contained 6 identical subunits . No high molecular weight form of GDH present in crude extracts was found after elution of the enzyme from a 5'AMP-Sepharose column with 4 M urea . The purified enzyme functions in both directions i.e . amination and deamination and is strictly specific for NAD . 2-Oxo glutarate, glutamate or 2-mercaptoethanol protects against heat inactivation . NADH or ammonia, on the other hand, makes GDH more sensitive to heat . The purified enzyme undergoes thermal inactivation process. Mol Cell Biol, 1985 Jun, 5(6), 1260 - 7 Gene amplification in Tetrahymena thermophila: formation of extrachromosomal palindromic genes coding for rRNA; Yao MC et al.; Tetrahymena thermophila contains in the macronucleus multiple copies of extrachromosomal palindromic genes coding for rRNA (rDNA) which are generated from a single chromosomal copy during development . In this study we isolated the chromosomal copy of rDNA and determined the structure and developmental fate of the sequence surrounding its 5' junction . The result indicates that specific chromosomal breakage occurs at or near the 5' junction of rDNA during development . The breakage event is associated with DNA elimination and telomeric sequence addition . Similar results were also found previously for the 3' junction of this gene . These results could explain how the extrachromosomal rDNA is first generated . Near both junctions of the chromosomal rDNA, a pair of 20-nucleotide repeats was found . These sequences might serve as signals for site-specific breakage . In addition, we found a pair of perfect inverted repeats at the 5' junction of this gene . The repeats are 42 nucleotides long and are separated by 28 nucleotides . The existence of this structure provides a simple explanation for the formation of the palindromic rDNA. Anal Biochem, 1985 Jun, 147(2), 419 - 27 A cellulase assay coupled to cellobiose dehydrogenase; Canevascini G; An assay for cellulase activity based on the oxidation of cellobiose, formed during the cellulase reaction, with ferricyanide and a cellobiose dehydrogenase derived from the cellulolytic fungus Sporotrichum (Chrysosporium) thermophile is presented . Due to the restricted specificity of this enzyme for cellobiose and cellodextrins, glucose, which may be formed by the action of some cellulolytic components or by beta-glucosidase, does not contribute to the result . The negative interference of beta-glucosidase may be eliminated by glucono-delta-lactone inhibition . The assay, which is not influenced by cellobiose back-inhibition of the cellulase reaction, like the usual cellulase tests based on the increase in reducing power, is basically unspecific with respect to endo- or exo-acting enzymes giving rise to a total cellulase activity . With the use of an amorphous cellulose substrate (reprecipitated cellulose after dissolving in concentrated phosphoric acid), unpredictable effects due to cooperativity between endo- and exo-enzyme components were eliminated . An analytical procedure giving a linear response between activity and enzyme concentration and between activity and time of incubation has been worked out. J Cell Sci, 1985 Jun, 76, 179 - 87 Infection and maintenance of Holospora obtusa, a macronucleus-specific bacterium of the ciliate Paramecium caudatum; Fujishima M et al.; The gram-negative bacterium Holospora obtusa is a macronucleus-specific symbiont of the ciliate Paramecium caudatum, which invades the host cell via a food vacuole, infects its macronucleus and grows exclusively in the nucleus . From infection experiments, we showed that a property of the macronucleus that is necessary for it to be recognized and infected by H . obtusa is commonly provided by P . caudatum, P . multimicronucleatum and 14 species of the P . aurelia complex, but not by P . jenningsi, P . bursaria, P . trichium, P . duboscqui, Didinium nasutum, Blepharisma japonicum, Pseudourostyla levis, seven species of Euplotes or Tetrahymena thermophila . Furthermore, it was also shown that the bacteria that infect the macronuclei of P . multimicronucleatum and the P . aurelia species complex always disappear from the nuclei within 5 days and the infected bacteria are maintained stably in the host nuclei in only 13 out of 22 strains of P . caudatum . The results indicate that the species specificity of the habitat of H . obtusa is not simply a matter of its ability to penetrate the host nuclear membrane but depends on unknown factors that exist only in certain strains of P . caudatum. Cell, 1985 Jun, 41(2), 541 - 51 A high affinity topoisomerase I binding sequence is clustered at DNAase I hypersensitive sites in Tetrahymena R-chromatin; Bonven BJ et al.; Topoisomerase I is associated with DNAase I hypersensitive sites in the nontranscribed spacers flanking the rRNA genes in Tetrahymena thermophila . The endogenous topoisomerase I introduces site and strand specific single-strand cleavages in the rDNA spacers in situ . The cleavages occur base specifically within a hexadecameric sequence element present in two or three direct repeats at the hypersensitive sites . The sequence specificity and polarity of the cleavage reaction are identical when the enzyme is reacted with naked rDNA, indicating that the repetitive element functions as a high-affinity topoisomerase I attraction site in the r-chromatin . The biological mechanism associated with this phenomenon appears to be widespread among eukaryotes, since the topoisomerase I recognition sequence is conserved in the rDNA spacers of phylogenetically remote organisms, such as fungi, slime molds, ciliates, and insects. Biochim Biophys Acta, 1985 May 31, 807(3), 293 - 9 Photoaffinity labeling of the TF1-ATPase from the thermophilic bacterium PS3 with 3'-O-(4-benzoyl)benzoyl ADP; Bar-Zvi D et al.; 3'-O-(4-Benzoyl)benzoyl ADP (BzADP) was used as a photoaffinity label for covalent binding of adenine nucleotide analogs to the nucleotide binding site(s) of the thermophilic bacterium PS3 ATPase (TF1) . As with the CF1-ATPase (Bar-Zvi, D . and Shavit, N . (1984) Biochim . Biophys . Acta 765, 340-356) noncovalently bound BzADP is a reversible inhibitor of the TF1-ATPase . BzADP changes the kinetics of ATP hydrolysis from noncooperative to cooperative in the same way as ADP does, but, in contrast to the effect on the CF1-ATPase, it has no effect on the Vmax . In the absence of Mg2+ 1 mol BzADP binds noncovalently to TF1, while with Mg2+ 3 mol are bound . Photoactivation of BzADP results in the covalent binding of the analog to the nucleotide binding site(s) on TF1 and correlates with the inactivation of the ATPase . Complete inactivation of the TF1-ATPase occurs after covalent binding of 2 mol BzADP/mol TF1 . Photoinactivation of TF1 by BzADP is prevented if excess of either ADP or ATP is present during irradiation . Analysis by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate of the Bz{3H}ADP-labeled TF1-ATPase shows that all the radioactivity is incorporated into the beta subunit. J Biol Chem, 1985 May 25, 260(10), 6334 - 40 The nucleotide sequence of the 17S ribosomal RNA gene of Tetrahymena thermophila and the identification of point mutations resulting in resistance to the antibiotics paromomycin and hygromycin; Spangler EA et al.; We have determined the complete sequence of the nuclear gene encoding the small subunit (17 S) rRNA of the ciliated protozoan Tetrahymena thermophila . The gene encodes an RNA molecule which is 1753 nucleotides in length . The sequence of the Tetrahymena small subunit rRNA is homologous to those of other eukaryotes, and the predicted secondary structure for the molecule includes features which are characteristic of eukaryotic small subunit rRNAs . We have also determined the nature of two different mutations in the Tetrahymena 17 S gene which result in resistance to the aminoglycoside antibiotics paromomycin and hygromycin . In each case we have identified a single base change near the 3' end of the rRNA, within a region that is highly evolutionarily conserved in both sequence and secondary structure . Analysis of the effects of these mutations on rRNA structure, and of the impact of these drugs on translation, should help to elucidate the role of the small subunit ribosomal RNA in ribosome function. Eur J Biochem, 1985 May 15, 149(1), 41 - 6 DNA polymerases from the extremely thermophilic bacterium Thermus thermophilus HB-8; Ruttimann C et al.; Three DNA polymerase isoenzymes, which have been called A, B and C, were purified from Thermus thermophilus HB-8 . These enzymes can be separated by chromatography (pH 7.5) on phosphocellulose and DNA-agarose . Their relative molecular masses, as determined by glycerol gradient centrifugation, fall in the range of 110000-120000 . The three of them are devoid of exonuclease activity . Species A, B and C differ in their sensitivity towards N-ethylmaleimide (A greater than B greater than C) and urea (A greater than B = C) and also in their stability at high temperature (90 degrees C) (B greater than C greater than A) . In addition, these enzymes can be distinguished utilizing various templates under different conditions . Thus, with activated DNA and Mg2+ as a cofactor, the highest incorporation is obtained at 50 degrees C with enzyme A and at 63 degrees C with enzymes B and C . If Mg2+ is replaced by Mn2+, the optimal temperatures remain unchanged, but enzyme A is stimulated twofold, while the activities of enzymes B and C decrease to one-half . On the other hand, with either poly(dA) X (dT)10 or poly(dA-dT) and Mg2+, enzyme A is inactive and enzyme C is severalfold more active than enzyme B . With the former synthetic template, optimal temperatures are 50 degrees C (enzyme C) and 40 degrees C (enzyme B), while with poly(dA-dT) they both work best at 63 degrees C . In turn, only enzyme C is able to utilize poly(rA) X (dT)10, although only with Mn2+ as a cofactor. Biochim Biophys Acta, 1985 May 14, 815(2), 268 - 80 The effect of growth temperature on the thermotropic behavior of the membranes of a thermophilic Bacillus . Composition-structure-function relationships; Reizer J et al.; The following study was carried out with the aim of widening our understanding of the thermoadaptive mechanisms of the membrane of thermophiles, using Bacillus stearothermophilus var . nondiastaticus as test-organism . The phospholipids and their acyl chain composition of this Bacillus studied in relation to the physical properties of its membrane from bacteria grown at various temperatures . Phospholipids account for 68-75 weight% of the total lipid in cells grown at 45, 55 or 65 degrees C . Phosphatidylglycerol and diphosphatidylglycerol constitute up to 90% of the total phospholipids; no amino phospholipids were found . Increasing the growth temperatures from 45 degrees to 65 degrees C caused an approximately 4-fold decrease in the proportion of the branched-chain fatty acids and a 2-fold increase in the amount of the saturated acyl chains . The reduced proportion of the branched fatty acids was mainly due to a decrease in their anteiso forms . Unsaturated fatty acids were not produced by cells grown at 65 degrees C . In accordance with the fatty acid composition, the molecular packing of phospholipids in monolayers was more expanded with phospholipids from 45 degrees C grown cells as compared with cultures grown at 55 degrees C . The thermotropic gel to liquid-crystalline phase transition of the membrane lipids was monitored by differential scanning calorimetry and fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene . With increase of the growth temperature the phase transition was progressively shifted to higher but narrower range of temperatures . Completion of the lipid melting occurred always at temperatures below those employed for growth . A constructed phase diagram enabled to relate the growth temperature, the fatty acid composition and the lipid apparent microviscosity at temperatures not used in the present study for growth of the thermophile . The minimum temperature for growth and the upper boundary temperature of the least saturated lipid crystallization were extrapolated in this manner; they correspond to the experimentally determined minimal growth temperature . The apparent microviscosity, a measure of membrane order, decreased gradually and conspicuously as the growth temperature was elevated . The delimiting apparent microviscosity values, at the maximal (65 degrees C) and minimal (41 degrees C) growth temperatures were 0.8 and 1.8 poise, respectively . This lack of rigorous homeostatic control of the bulk lipid viscosity prompted reevaluation of the physiological significance of 'homeoviscous adaptation' in Bacillus stearothermophilus. J Biochem (Tokyo), 1985 May, 97(5), 1521 - 4 Thermophilic microspheres of peptide-like polymers and silicates formed at 250 degrees C; Yanagawa H et al.; We examined the possibility of chemical evolution in superheated hydrothermal environments and found the formation of microspheres at 250 degrees C and above from a mixture of glycine, alanine, valine, and aspartic acid . The microspheres did not form at lower temperatures and consisted of silicates and peptide-like polymers that contained imide bonds and amino acid residues having an abundance of valine . The results show the possibility of thermophilic cellular structures, which might be adopted by the extremely thermophilic organisms, if they exist, reported by Baross and Deming. Exp Cell Res, 1985 May, 158(1), 244 - 56 Effect of the antitubulin drug nocodazole on meiosis and postmeiotic development in Tetrahymena thermophila . Induction of achiasmatic meiosis; Kaczanowski A et al.; Nocodazole (ND), a potent antitubulin drug, can be used to dissect the steps of meiosis in Tetrahymena, presumably by interfering with the assembly of microtubules . Its effects depend upon the time during conjugation at which the drug is applied . When applied prior to the elongation of the micronucleus into the characteristic 'crescent' configuration, no crescent is formed and the chromosomes of prepachytene and pachytene condense into spherical nuclei . If ND is applied after micronuclear elongation has begun, but before it is fully elongated, the chromosomes fail to synapse and appear in metaphase I as unpaired monovalents . In contrast, the metaphase I chromosomes appear as bivalents when ND is applied later, during or after the crescent has reached its maximum elongation . Still later, application of ND inhibits chromosome movements during anaphase and telophase of either meiotic division, but does not prevent separation of kinetochores . In some of the blocked restitutive nuclei an additional round of chromosome replication occurs, corresponding to the third pregamic division in normal conjugation . The hyperploid micronuclei produced by such treatment may be useful in certain genetic manipulations and in studying the regulation of nuclear DNA content. Exp Cell Res, 1985 May, 158(1), 159 - 69 Deciliation interferes with cell-cycle progression in Tetrahymena; Seyfert HM et al.; The impact of ciliary regeneration upon cell-cycle progression of the ciliate Tetrahymena was studied . It was found that cell division ceases during ciliary regeneration, and starts again about 4 h after deciliation . Deciliation of an asynchronously multiplying culture results in a rapid interruption of DNA synthesis, followed by resumption 1 h later . This was shown by pulse-labelling the cells with {3H}thymidine at various times after deciliation . Cytophotometric determinations of the macronuclear DNA content substantiated these observations, since the average DNA content per cell remained constant within the first hour of regeneration, confirming the labelling experiments, after which it rose . At its maximum, the average DNA content was more than doubled as compared with the beginning of the experiment . This indicates that a substantial proportion of the regenerating cells performed two rounds of DNA replication prior to cell division . The massive drop in the average DNA content during the fifth hour after deciliation indicates that the culture becomes partly synchronized for cell division by the deciliation procedure . The division synchrony results from a greater delay of the next cell division when G2 cells are deciliated than occurs in G1 cells . This was shown by deciliating cultures of Tetrahymena thermophila cells in the respective stages of the cell cycle, which had been partly synchronized by elutriator centrifugation . Thus, deciliation followed by ciliary regeneration causes a varying degree of retardation in progression through the cell cycle, being greatest for G2 cells and least for G1 cells. Chest, 1985 May, 87(5), 658 - 61 Pyridoxine deficiency in children treated with isoniazid; Pellock JM et al.; Isoniazid-induced deficiency of pyridoxine (vitamin B6) is reportedly not uncommon in adults but rare in children . In the present study, 38 children had serum levels of pyridoxine tested while receiving therapy with isoniazid . A biologic assay using the protozoan Tetrahymena thermophila determined pyridoxine status after 2 to 18 months of therapy with isoniazid . Five children (13 percent) were deficient . None had definitive clinical symptoms or signs consistent with pyridoxine deficiency . Three had normal nerve conduction velocity . Children receiving isoniazid in dosages greater than 10 mg/kg/day had a higher incidence of deficiency . Present recommendations for withholding pyridoxine prophylaxis from children receiving isoniazid therapy must be reconsidered in light of these findings, particularly in those children who are debilitated or have a poor nutritional history with a known pyridoxine deficit prior to therapy with isoniazid. Mutat Res, 1985 May, 145(3), 157 - 64 Repair of 8-methoxypsoralen induced DNA interstrand cross-links in Tetrahymena thermophila . The effect of inhibitors of macromolecular synthesis; Nielsen PE et al.; The effect of several growth-inhibiting compounds on the repair of 8-methoxypsoralen-UVA-light-induced DNA interstrand cross-links has been studied in the protozoan Tetrahymena thermophila . The repair process was analyzed by the alkaline elution technique and could be divided into 3 phases: a protein-DNA complexing phase, a DNA-incision phase and finally a DNA-ligation phase . The incision was found to be completely inhibited by novobiocin (50 micrograms/ml), nalidixic acid (150 micrograms/ml), n-butyrate (15 mM) and cycloheximide (1 microgram/ml), while no effect was observed for cytosine-1-beta-D-arabinofuranoside (10 mM), puromycin (1 mM), hydroxyurea (5 mM) or 3-aminobenzamide (2.5 mM) . None of the compounds showed any effect on the protein-DNA complexing step, and the ligation was partly inhibited only by nalidixic acid (150 micrograms/ml) . The involvement of topoisomerases in the repair of psoralen-induced DNA interstrand cross-links is suggested. J Bacteriol, 1985 May, 162(2), 768 - 72 Buffering capacity of bacilli that grow at different pH ranges; Krulwich TA et al.; Cytoplasmic buffering capacities and buffering by whole cells were examined in six bacterial species: Bacillus acidocaldarius, Bacillus stearothermophilus, Escherichia coli, Bacillus subtilis, Bacillus alcalophilus, and Bacillus firmus RAB . Acid-base titrations were conducted on whole cells and cells permeabilized with Triton X-100 or n-butanol . In all of the species examined, the buffering capacity of intact cells was generally a significant proportion of the total buffering capacity, but the magnitude of the buffering capacity varied from species to species . Over the entire range of pH values from 4 to 9.5, B . subtilis exhibited a cytoplasmic buffering capacity that was much higher than that of B . stearothermophilus, B . acidocaldarius, or E . coli . The latter three species had comparable cytoplasmic buffering capacities at pH 4 to 9.5, as long as optimal conditions for cell permeabilization were employed . All of the nonalkalophiles exhibited a decrease in cytoplasmic buffering capacity as the external pH increased from pH 5 to 7 . At alkaline pH values, the two thermophiles in the study had particularly low cytoplasmic buffering capacities, and the two alkalophilic bacteria had appreciably higher cytoplasmic buffering capacities than any of the other species studied . Cytoplasmic buffering capacities as high as 1,100 nmol of H+ per pH unit per mg of protein were observed in alkalophilic B . firmus RAB . Since previous studies have shown that immediate cytoplasmic alkalinization occurs upon loss of the active mechanisms for pH homeostasis in the alkalophiles, the very high buffering capacities apparently offer no global protection of internal pH . Perhaps, the high buffering capacities reflect protective mechanisms for specific macromolecules or process rather than part of the mechanisms for bulk pH homeostasis. Dev Biol, 1985 May, 109(1), 1 - 8 Modulation of linker histones during development in Tetrahymena: selective elimination of linker histone during the differentiation of new macronuclei; Chicoine LG et al.; Macronuclei of Tetrahymena thermophila contain a typical H1 which has been shown to be missing from micronuclei . Instead, micronuclei contain three unique polypeptides, alpha, beta, and gamma, which are associated with linker regions of micronuclear chromatin . In this report polyclonal antibodies raised against macronuclear H1 are shown to react with alpha, beta, and gamma by immunoblotting analyses . This result suggests that these polypeptides share some common structural feature(s) . Also consistent with this result is the finding that both macro- and micronuclei in growing and mating cells stain positively with H1 antibodies by in situ indirect immunofluorescence . However, these analyses demonstrate that the level of linker histone is greatly reduced in the micronucleus of starved cells and in young macronuclear anlagen . These results are in agreement with earlier biochemical studies and together provide strong evidence that dramatic changes in linker histone accompany nuclear differentiation (and dedifferentiation) in Tetrahymena. Mycopathologia, 1985 May, 90(2), 81 - 3 Amylase and growth characteristics of Papulaspora thermophilia; Adams PR; Mycelial dry weight of Papulaspora thermophilia reached a maximum of about 96 mg after six days of growth in a starch-yeast medium . Extracellular amylase activity was not measurable during this growth period and remained thereafter only about 0.1 unit per ml for 30 days, yet starch concentration reduced rapidly, and reducing sugar appeared in the extracellular medium within the first few days of incubation. Biochem Biophys Res Commun, 1985 Apr 30, 128(2), 781 - 7 Amino acid sequence of cytochrome c-552 from Thermus thermophilus HB8; Titani K et al.; The complete amino acid sequence of thermophilic cytochrome c-552 from Thermus thermophilus HB8 is presented . The 131-residue sequence was derived by analysis of three cyanogen bromide fragments of the S-carboxymethylated apo-protein and their subpeptides . The sequence is homologous to c-type cytochromes, especially in the heme-binding region. Biochim Biophys Acta, 1985 Apr 29, 828(3), 369 - 74 Quarter field resonance and integer-spin/half-spin interaction in the EPR of Thermus thermophilus ferredoxin . Possible new fingerprints for three iron clusters; Hagen WR et al.; We describe two new characteristics of the EPR of the seven-iron containing ferredoxin from Thermus thermophilus . First, the reduced state of the 3Fe center, which has traditionally been considered to be EPR-silent, has been found to exhibit a delta m = 4 transition, which is unique for Fe-S centers . This signal is similar to that of high-spin Fe2+-EDTA and supports the suggestion that the ground electronic state of the 3Fe cluster is S = 2 . Second, we have recorded the EPR spectrum of the fully reduced protein at 9 and 15 GHz and found that changes occur in the signal which are consistent with a weak electronic spin-spin interaction between the {4Fe-4S}+ (S = 1/2) and the reduced 3Fe center . A theoretical explanation is given for the observation of interaction signals with constant effective g values. Nucleic Acids Res, 1985 Apr 11, 13(7), 2661 - 80 Conserved arrangements of repeated DNA sequences in nontranscribed spacers of ciliate ribosomal RNA genes: evidence for molecular coevolution; Challoner PB et al.; We have analyzed the nucleotide sequences of the nontranscribed spacer (NTS) and transcription initiation and termination regions of the extrachromosomal rDNAs of the ciliated protozoans Tetrahymena thermophila and Glaucoma chattoni . The sequences surrounding the sites of transcription initiation and termination are highly conserved . The only extensive homologies of the NTS regions occur in five sets of dispersed repetitive sequences . Type I, II and III repeats in the 5' NTS are strongly conserved in sequence between Tetrahymena and Glaucoma in the case of the type I and III repeats, and in location relative to the transcription initiation site in the case of type I and II repeats . We identify two new repeat types, designated IV and V, in the 3' NTS . The sequence of type IV repeats, and the location relative to the transcription termination site of type IV and V repeats, are conserved . All five types of repeats are interspersed with nonconserved DNA sequences . These results suggest that the five repeat types in the 5' and 3' NTSs are important in rRNA gene function; the sequence organization, and the differing rates of divergence between species of the repeat types, provide strong evidence for their functional selection through the process of molecular coevolution. Nucleic Acids Res, 1985 Apr 11, 13(7), 2647 - 59 Autonomously replicating sequences from the non transcribed spacers of Tetrahymena thermophila ribosomal DNA; Amin AA et al.; The non-transcribed spacer (NTS) regions of the linear extrachromosomal palindromic rDNA from the ciliated protozoan Tetrahymena thermophila contain, in addition to sequences regulating transcription, the origin(s) of bidirectional replication as well as telomere associated sequences . These NTS regions function as autonomous replication sequences (ARS) in the yeast Saccharomyces cerevisiae (Kiss, G.B., Amin, A.A . and Pearlman, R.E., Mol . Cell Biol . 1: 535-543, 1981) . We have now identified in the 5' NTS two adjacent but non-overlapping restriction fragments which function as ARS in yeast . These fragments contain 200 bp of duplicated sequence and include the in vivo origin of rDNA replication . The ARS in the 3' NTS has been subdivided into a sequence which allows high frequency transformation of yeast but with transformants extremely unstable when grown either under selective or non-selective conditions, and a sequence which appears to be required for plasmid maintenance yet not to be essential for high frequency transformation . Detailed analysis of the DNA sequences in these regions gives rise to interesting information about structural and functional features of the molecule. Microbiologica, 1985 Apr, 8(2), 191 - 6 Chemoresistances among 80 Campylobacter strains isolated from childhood gastroenteritis cases; Figura N et al.; The antimicrobial susceptibility of 80 thermophilic Campylobacter strains, 61 C . jejuni and 19 C . coli, isolated from childhood gastroenteritis cases has been studied . Gentamicin and chloramphenicol were effective against all strains; beta-lactams, except carbenicillin and ticarcillin, had on the whole little activity . 26.2% of strains proved to be resistant to tetracycline and 7.5% to erythromycin; erythromycin-resistance was found significantly more often in the species C . coli and always associated to clindamycin-resistance . The high prevalence of strains resistant to erythromycin suggests chloramphenicol and gentamicin as possible alternative drugs in the treatment of life-threatening Campylobacter infections. J Clin Microbiol, 1985 Apr, 21(4), 553 - 7 Thermophilic bacteria: a new cause of human disease; Rabkin CS et al.; We studied a group of 31 bacterial isolates from clinical specimens, received by the Centers for Disease Control since 1961, which have been denoted thermophilic for their unusual ability to grow at 50 degrees C . Microbiological characteristics were determined for the group, and an assessment of their clinical significance was made based on retrospective chart review . These bacteria are all gram-negative, nonfermentative, nonsporulating rods, most of which grow better at 42 or 50 degrees C than at 35 degrees C . Some of the bacteria could be implicated as the etiological agents for meningitis, endocarditis, and septicemia . Thermophilic bacteria should be considered potential pathogens when isolated from appropriate clinical specimens. J Bacteriol, 1985 Apr, 162(1), 102 - 5 Sequence of a cellulase gene of the thermophilic bacterium Clostridium thermocellum; Beguin P et al.; The nucleotide sequence of the celA gene, encoding the extracellular endoglucanase A of Clostridium thermocellum, was determined and compared with the NH2-terminal amino acid sequence of the purified enzyme . The mature protein appeared to be extended by a signal sequence of 32 amino acids . A segment of 23 amino acids was duplicated at the COOH-terminal end of the protein . The putative GUG initiation codon was preceded by an AGGAGG sequence, typical of procaryotic ribosomal binding sites . The segment of DNA presumably specifying transcriptional initiation contained a high percentage of adenine and thymine residues, including an adenine-thymine tract extending over 54 base pairs. Exp Cell Res, 1985 Apr, 157(2), 315 - 21 A transmissible developmental block in Tetrahymena thermophila; Kaney AR; When the amicronucleate mutant BI3840 of Tetrahymena thermophila is mated with normal micronucleate cells, it receives a pronucleus from its partner but there is no further nuclear development and the conjugants separate, retaining their original macronuclei . Both of these sexually mature exconjugants and any cells with which they are mated show an unconditional block in macronuclear development . Although prezygotic nuclear divisions, nuclear transfer and post-zygotic nuclear divisions appeared normal upon cytological analysis of Giemsa-stained conjugants, macronuclear development was invariably aborted . Since the original macronucleus was resorbed, the cells were rendered amacronucleate and they died . When no macronuclear development was initiated, as in crosses with the aneuploid strain A* (III), the exconjugants were viable and retained their original macronuclei . This pattern was invariant with three different strains serving as the original micronuclear source, and in the case of one non-BI3840 exconjugant, persisted for over 200 cell generations . Exconjugants from a cross of one of the micronuclear donors with strain A* (III) did not show arrested development when crossed . It thus appears likely that there is conjugal transfer of non-nuclear information originating in BI3840 which is self-replicating and which causes an arrest in macronuclear development. Can J Microbiol, 1985 Apr, 31(4), 339 - 45 Isolation and characterization of antibiotic-resistance plasmids in thermophilic bacilli; Hoshino T et al.; Four antibiotic-resistance plasmids isolated from thermophilic bacilli were characterized in detail . Three tetracycline-resistance (Tc1) plasmids were designated as pTHT9 (7.7 kilobases (kb}, pTHT15 (4.5 kb) and pTHT22 (8.4 kb) . From the results of restriction endonuclease analysis and the subsequent Southern hybridization, these were found to possess extensive genetic homology in the regions that include the replication origin and the Tcr gene . Detailed restriction maps of the smallest Tcr plasmid pTHT15 and a kanamycin-resistance (Kmr) plasmid pTHN1 (4.8 kb) were constructed . The positions of antibiotic-resistance loci and regions essential for plasmid replication were determined by cloning plasmid fragments in Bacillus subtilis . These four plasmids were found to replicate and express the resistance genes stably in both B . subtilis and B . stearothermophilus. Proc Natl Acad Sci U S A, 1985 Apr, 82(8), 2452 - 5 An unusual genetic code in nuclear genes of Tetrahymena; Horowitz S et al.; We have cloned and partially sequenced two histone H3 genes of Tetrahymena thermophila . The DNA sequences strongly suggest that both genes are active in the vegetatively growing cell . Comparison of the derived amino acid sequences of these two genes with the actual sequence of Tetrahymena histone H3 results in the surprising conclusion that TAA codes for glutamine . This represents the first demonstration of a coding function for this termination codon of the "universal" code . This observation has important implications for the evolution of ciliates and of the genetic code. Biochem Int, 1985 Apr, 10(4), 655 - 62 Isolation and partial characterization of BstVI, a thermostable isoschizomer of XhoI; Vasquez C; A type II restriction endonuclease, which has been named BstVI, was isolated and partially purified from a spore-forming, gram-positive thermophilic bacilli . On the basis of its digestion patterns on various DNA's, it was concluded that this enzyme is an isoschizomer of XhoI, isolated originally from Xanthomonas holcicola . Besides being highly thermostable, the enzyme is produced in very large amounts by this bacterial strain . A single purification step renders it free of unspecific nucleases and suitable for performing restriction analysis and cloning experiments. Nucleic Acids Res, 1985 Mar 25, 13(6), 1871 - 89 Sequence requirements for self-splicing of the Tetrahymena thermophila pre-ribosomal RNA; Price JV et al.; The sequence requirements for splicing of the Tetrahymena pre-rRNA have been examined by altering the rRNA gene to produce versions that contain insertions and deletions within the intervening sequence (IVS) . The altered genes were transcribed and the RNA tested for self-splicing in vitro . A number of insertions (8-54 nucleotides) at three locations had no effect on self-splicing activity . Two of these insertions, located at a site 5 nucleotides preceding the 3'-end of the IVS, did not alter the choice of the 3' splice site . Thus the 3' splice site is not chosen by its distance from a fixed point within the IVS . Analysis of deletions constructed at two sites revealed two structures, a hairpin loop and a stem-loop, that are entirely dispensable for IVS excision in vitro . Three other regions were found to be necessary . The regions that are important for self-splicing are not restricted to the conserved sequence elements that define this class of intervening sequences . The requirement for structures within the IVS for pre-rRNA splicing is in sharp contrast to the very limited role of IVS structure in nuclear pre-mRNA splicing. J Biol Chem, 1985 Mar 25, 260(6), 3251 - 4 Evidence for N coordination to Fe in the {2Fe-2S} clusters of Thermus Rieske protein and phthalate dioxygenase from Pseudomonas; Cline JF et al.; Rieske-type iron/sulfur proteins and several NADH-dependent oxygenases contain Fe/S clusters with similar spectral and magnetic properties . Purified Rieske iron/sulfur protein from Thermus thermophilus contains two apparently identical {2Fe-2S} clusters in a polypeptide having only four cysteine residues, and it has been proposed that each Fe/S cluster is coordinated to two cysteine S-atoms and to an unknown number of other non-sulfur atoms (Fee, J . A., Findling, K . L., Yoshida, T., Hille, R., Tarr, G . E., Hearshen, D . O., Dunham, W . R., Day, E . P., Kent, T . A., and Munck, E . (1984) J . Biol . Chem . 259, 124-133) . We have examined the Rieske protein from Thermus and the phthalate dioxygenase from Pseudomonas cepacia with electron nuclear double resonance (ENDOR) and pulsed EPR methods and report here evidence for the direct coordination of nitrogenous ligands to the Fe/S clusters in these proteins . The electron nuclear double resonance signals arising from 14N have been interpreted in terms of a strongly coupled ligand with AN = approximately 26-28 MHz and a weakly coupled ligand with AN = approximately 9 MHz . The pulsed EPR spectrum shows a rich pattern of lines in the Fourier transformed data having peaks in the range of 0.8 to 6.7 MHz . The lower frequency resonances are tentatively associated with coupling of the unpaired spin to the remote N-atoms of coordinated imidazole rings. Eur J Biochem, 1985 Mar 15, 147(3), 503 - 10 Conserved unpaired adenine residues are important for ordered structures of 5S ribosomal RNA . An infrared study of the secondary and tertiary structure of Thermus thermophilus 5S rRNA; Bohm S et al.; An improved set of infrared calibration spectra for the determination of G X C and A X U base pairs leads to 32 +/- 3 G X C (+ G X U) and 4 +/- 1 A X U base pairs for Thermus thermophilus 5S RNA in the presence and absence of Mg2+ . These results give further support for the consensus secondary structure of 5S RNA recently proposed by several groups . T . thermophilus 5S RNA shows, in the presence of Mg2+, a distinct two-step thermal melting of its ordered structure . Based on new data about the stacking dependence of infrared intensities of unpaired ribonucleotides the spectral changes of the low-temperature transition should be explained by melting of stacked arrangements of unpaired bases and/or non-standard base pairs . Striking is the reduction in A stacking, which is not related to the melting of A X U base pairs, indicating the importance of the mostly conserved unpaired adenines for the Mg2+ stabilized higher-order structures especially within internal loops of 5S RNA. Biochemistry, 1985 Mar 12, 24(6), 1476 - 83 Spectral properties and function of two lumazine proteins from Photobacterium; Lee J et al.; The spectral properties are compared for two 6,7-dimethyl-8-ribityllumazine proteins from marine bioluminescent bacteria, one from a psychrophile, Photobacterium phosphoreum, and the other from a thermophile, Photobacterium leiognathi . The visible spectral properties, which are the ones by which the protein performs its biological function of bioluminescence emission, are almost the same for the two proteins: at 2 degrees C and 50 mM Pi, pH 7, fluorescence quantum yield phi F = 0.59 and 0.54, respectively; fluorescence lifetime tau = 14.4 and 14.8 ns, respectively; fluorescence maxima, both 475 nm; absorption maximum, 417 and 420 nm, respectively; circular dichroism minima at around 420 nm, both -41 X 10(3) deg cm2 dmol-1 . The ligand binding sites therefore must provide very similar environments, and arguments are presented that the bound ligand is relatively exposed to solvent . The dissociation equilibrium was studied by steady-state fluorescence polarization . The thermophilic protein binds the ligand with Kd (20 degrees C) = 0.016 microM, 10 times more tightly than the other protein {Kd (20 degrees C) = 0.16 microM} . The origin of the binding difference probably resides in differences in secondary structure . The tryptophan fluorescence spectra of the two proteins are different, but more significant is an observation of the decay of the tryptophan emission anisotropy . For the psychrophilic lumazine protein this anisotropy decays to zero in 1 ns, implying that its single tryptophan residue lies in a very "floppy" region of the protein . For the other protein, the anisotropy exhibits both a fast component and a slow one corresponding to rotation of the protein as a whole.(ABSTRACT TRUNCATED AT 250 WORDS) Biochemistry, 1985 Mar 12, 24(6), 1467 - 75 Chemical characterization of lumazine protein from Photobacterium leiognathi: comparison with lumazine protein from Photobacterium phosphoreum; O'Kane DJ et al.; The properties of lumazine proteins purified from the marine bioluminescent bacteria Photobacterium phosphoreum, a psychrophile, and Photobacterium leiognathi, a relatively thermophilic species, are compared . An accurate 1:1 stoichiometry of binding of the ligand 6,7-dimethyl-8-ribityllumazine to each lumazine protein is established by back-titration of the apoprotein with the authentic ligand, using both fluorescence and absorption measurements . Neither protein contains metal cofactors, organic phosphorus, or carbohydrate . Both proteins are anionic and hydrophilic . They each contain a single Trp residue and have blocked amino terminals but otherwise differ in amino acid composition and other properties (P . phosphoreum and P . leiognathi, respectively): Met (internal), 1, 2; Cys, 2, 1; Arg, 4, 7; pI, 4.78 and 4.83, 4.38 and 4.45; Mr, 19 750, 21 300 . In the P . phosphoreum protein both Cys residues are accessible, but in the P . leiognathi protein the single Cys is "buried" . Modification of this buried Cys and at least one Cys in the P . phosphoreum protein prevents binding of the ligand . The UV and visible absorption spectra of both lumazine proteins denatured in 6 M guanidine hydrochloride can be accurately modeled by using the number of equivalents of the lumazine derivative and blocked aromatic amino acid model compounds determined by chemical and spectrophotometric analyses for Trp, Tyr, and Phe. Nucleic Acids Res, 1985 Mar 11, 13(5), 1543 - 57 Topoisomerase I has a strong binding preference for a conserved hexadecameric sequence in the promoter region of the rRNA gene from Tetrahymena pyriformis; Andersen AH et al.; Topoisomerase I is in situ associated with DNaseI hypersensitive sites located in the promotor and terminator regions of the extrachromosomal rDNA in Tetrahymena thermophila at sites with sequences fitting the motif (sequence in text) Reconstitution experiments with purified topoisomerase I and cloned fragments of rDNA demonstrate that the enzyme exhibits the same binding and cleavage properties on naked DNA . These observations are striking as topoisomerase I previously has been found to exhibit low sequence specificity . The specific binding of the enzyme has an absolute requirement for divalent cations with a preference for Ca2+ . The strong binding to the hexadecamer has been characterized by competition experiments, and it has been used to determine the molecular weight of the enzyme. J Biochem (Tokyo), 1985 Mar, 97(3), 899 - 909 Fructose 1,6-bisphosphate-dependent L-lactate dehydrogenase from Thermus aquaticus YT-1, an extreme thermophile: activation by citrate and modification reagents and comparison with Thermus caldophilus GK24 L-lactate dehydrogenase; Machida M et al.; Heat-stable fructose 1,6-bisphosphate-dependent L-lactate dehydrogenase {EC 1.1.1.27} was purified from an extremely thermophilic bacterium, Thermus aquaticus YT-1 . The amino acid composition and NH2-terminal 34 amino acid sequence of the enzyme were determined . Its NH2-terminal sequence shows high homology with those of Thermus caldophilus GK24 (82% identity) and some other bacterial L-lactate dehydrogenases (44-53% identity), indicating the close phylogenic relationship of the two Thermus species . At the same time, the two Thermus L-lactate dehydrogenases were found not to be identical not only chemically but also kinetically and immunologically . Citrate activated the T . aquaticus enzyme in the weak acidic pH region, while fructose 1,6-bisphosphate did in both acidic and neutral pH regions . The maximum activity obtained with citrate at pH 5.0 was about 2.5 times higher than that in the presence of fructose 1,6-bisphosphate at pH 6.7 . The enzymes modified with 2,3-butanedione, acetic anhydride and diethyl pyrocarbonate in the presence of both NADH and oxamate were desensitized to fructose 1,6-bisphosphate, and the modified enzymes were active even in the absence of fructose 1,6-bisphosphate . All of the modified enzymes examined were still activated by citrate similarly to the native enzyme . These results suggest that the mechanism of activation by citrate is different from that by fructose 1,6-bisphosphate, and that the citrate-binding site is different from the fructose 1,6-bisphosphate-binding site. Appl Environ Microbiol, 1985 Mar, 49(3), 724 - 6 Effect of seeding during thermophilic composting of sewage sludge; Nakasaki K et al.; The effect of seeding on the thermophilic composting of sewage sludge was examined by measuring the changes in CO2 evolution rates and microbial numbers . Although the succession of thermophilic bacteria and thermophilic actinomycetes clearly reflected the effect of seeding, no clear difference was observed in the overall rate of composting or quality of the composted product. J Bacteriol, 1985 Mar, 161(3), 981 - 8 Electron microscopy of the secondary structure in partially denatured rRNAs of Escherichia coli and Bacillus stearothermophilus; Klein BK et al.; Partially denatured 16S and 23S rRNAs from the thermophile Bacillus stearothermophilus show characteristic loop patterns when observed by electron microscopy . The patterns are very similar to those seen in rRNAs from Escherichia coli . At least 2 of 4 most stable interactions in 16S rRNA and 8 of 12 interactions in 23S rRNA are in common for the two species . These interactions correspond well to features of secondary structure in models inferred for rRNA from phylogenetic sequence comparisons and chemical modification studies . However, two additional large loops, enclosing large portions of the 23S rRNA, have been detected in B . stearothermophilus for the first time, and even though other loops are similar, their relative frequencies vary in the two species . Much of the variation is consistent with relative delta G degree values for putative base-paired stems at the base of different loops; but the 5'-terminal loops in 23S rRNA, for example, are unaccountably far less stable in B . stearothermophilus . Also, in general, structural features are not differentially stabilized in B . stearothermophilus; the relative stability of secondary structure in its ribosomes at elevated growth temperatures must involve interactions with ribosomal proteins or other cellular components. Int J Radiat Biol Relat Stud Phys Chem Med, 1985 Mar, 47(3), 291 - 7 Membrane damage and its repair in the thermophilic bacterium, Thermus thermophilus HB-8 exposed to u.v . radiation and 60Co gamma-rays; Suzuki S; When exposed to either u.v . radiation or 60Co gamma-rays, the thermophilic bacterium, Thermus thermophilus HB-8, which can grow at 49-85 degrees C, lost its ability to take up extracellular K+ in a dose-dependent manner . However, the loss was reduced by incubation at 37 degrees C after exposure to u.v . radiation or gamma-rays . Cell survival after exposure to 60Co gamma-rays, as measured by colony formation, was increased by incubation at 37 degrees C after exposure, whereas cell survival after u.v . radiation was not . These results, therefore, indicate that the loss of ability of cells to take up K+ after u.v . radiation was not due to cell death but some damage to the membrane itself, and that the membrane damage can be repaired . Lipid peroxidation is not responsible for the membrane damage, because HB-8 cells do not contain unsaturated fatty acids in their membranes. J Inorg Biochem, 1985 Mar-Apr, 23(3-4), 279 - 88 Potentiometric study of cytochrome c1aa3 from Thermus thermophilus; Yoshida T et al.; We have examined the redox behavior of the cytochrome c1aa3 complex from Thermus thermophilus . In potentiometric titrations the cytochrome c behaves as an independent center having n = 1 and E = 205 mV (NHE) . Under the assumption that the individual centers equilibrate independently in this experiment, changes in the absorption band at 603 nm have been resolved into two components: cytochrome a (n = 1, Em = 270 mV, 60% spectral contribution) and cytochrome a3 (n = 2, Em = 360 mV, 40% spectral contribution) . The n = 2 process was attributed to strong chemical coupling between cytochrome a3 and CuB . The enzyme was also titrated with a mixture of NADH and PMS, and the results are shown not to conform to a model of intramolecular equilibrium according to the equilibrium constants obtained from the potentiometric titration . It is suggested that a conformational equilibrium within the complex may control electron transfer between cytochromes a and a3. Biol Chem Hoppe Seyler, 1985 Mar, 366(3), 223 - 31 Purification, amino-acid sequence and some properties of the ferredoxin isolated from Bacillus acidocaldarius; Schlatter D et al.; Ferredoxin was isolated from the aerobic, thermophilic and acidophilic bacterium Bacillus acidocaldarius and its sequence of 78 amino acids completely determined by automated Edman degradation of the protein and of peptides derived from chemical cleavage between aspartic acid and proline and from enzymatic digestions . The optical spectrum of the oxidized protein has a broad maximum around 400 nm . The ferredoxin is thermostable: its absorbance begins to decrease only at incubation over 71 degrees C . The number of iron and inorganic sulphur atoms per molecule was determined to be 5.3 and 5.0, respectively . The calculated molar extinction coefficient was 23 000 M-1 X cm-1, the molecular mass of the apoferredoxin 8 872 Da . Contrary to all expectations, the sequence of B . acidocaldarius ferredoxin shows very little homology to that of B . stearothermophilus but closely resembles that of Thermus thermophilus. Can J Biochem Cell Biol, 1985 Mar, 63(3), 153 - 61 Complexes of cytochrome caa3 from the thermophilic bacterium PS3 formed with ligands and during catalytic activity; Sone N et al.; PS3 (thermophilic bacterium) cytochrome caa3 reacts slowly with cyanide which forms a low-spin complex with the CuBa3 centre . Partial reduction under catalytic conditions increases the rate of cyanide binding, and the reaction constant is rather similar to that of the mammalian enzyme, but the partially reduced complex dissociates more rapidly than does the corresponding eukaryotic complex . A simple biphasic reaction can account for the results obtained . The azide complex of partially reduced PS3 cytochrome caa3 shows an alpha-peak blue shift similar to that of the mammalian enzyme . PS3 cytochrome caa3 forms an oxyferri ("oxygenated") species like the mammalian enzyme, but does not undergo high- to low-spin changes during the aerobic steady state with ascorbate and N,N,N',N'-tetramethyl-p-phenylenediamine as substrates . Interactions seen in cytochrome oxidase-ligand reactions and its spin-state changes are therefore intrinsic to the enzyme's large catalytic subunits and do not require the presence of the small nuclear-encoded subunits found in eukaryotic systems. Nucleic Acids Res, 1985 Feb 25, 13(4), 1399 - 412 DNA methylation in thermophilic bacteria: N4-methylcytosine, 5-methylcytosine, and N6-methyladenine; Ehrlich M et al.; While determining the minor and major base composition of the DNA from 17 types of thermophilic bacteria by high performance liquid chromatography (HPLC) of enzymatic digests, we have discovered a novel base, N4-methylcytosine (m4C) . Its structure was proven by comparison of the DNA-derived nucleoside to the analogous authentic compound by HPLC, UV spectroscopy, and mass spectroscopy . Eight of the bacterial DNAs contained m4C . Only two contained the common minor base, 5-methylcytosine (m5C), and neither of these was from an extreme thermophile . The other prevalent modified base of bacterial DNA, N6-methyladenine (m6A), was found in nine of the DNAs . Restriction analysis revealed that four of the DNAs had dam-type (Gm6ATC) methylation patterns . Due to the propensity of m5C residues to be deaminated by heat to thymine residues and to inefficient repair of the resulting mismatched base pairs, thermophiles with optimal growth temperatures of greater than or equal to 60 degrees C generally may avoid having m5C in their genomes . Instead, some of them have deamination-resistant m4C residues. J Biol Chem, 1985 Feb 25, 260(4), 2064 - 8 Differential selectivity of cholinephosphotransferase and ethanolaminephosphotransferase of Tetrahymena for diacylglycerol and alkylacylglycerol; Smith JD; The glycerophospholipids of the ciliate protozoan Tetrahymena thermophila differ greatly in their content of alkylacylglycerol with phosphatidylcholine, phosphatidylethanolamine, and 2-aminoethylphosphonolipid containing 60, 4, and 53% glyceryl ether, respectively . This difference is achieved by differences in the selectivities of cholinephosphotransferase (EC 2.7.8.2) and ethanolaminephosphotransferase (EC 2.7.8.1) for alkylacylglycerol and diacylglycerol . When the two enzymes are assayed in vitro using only endogenous diglyceride as substrate, the newly formed phosphatidylcholine contains 37% glyceryl ether, while the newly formed phosphatidylethanolamine contains 5% glyceryl ether . The ethanolaminephosphotransferase is stimulated equally well by addition of diolein and dipalmitin, but the diacylglycerols have no effect on the glyceryl ether content of phosphatidylethanolamine . In contrast, the glyceryl ether content of newly formed phosphatidylcholine decreases to 16% when the cholinephosphotransferase is exposed to diolein or dipalmitin . The ethanolaminephosphotransferase is not stimulated by addition of a 60:40 mixture of alkylacylglycerol/diacylglycerol . The cholinephosphotransferase is stimulated by the mixture to the same extent as it is by the diacylglycerols, with the glyceryl ether content of the newly formed phosphatidylcholine increasing to 52% . With the addition of alkylacylglycerol alone, the glyceryl ether content of the newly formed phosphatidylethanolamine increases to 10%, while that of the newly formed phosphatidylcholine increases almost to 60%. Nature, 1985 Feb 28-Mar 6, 313(6005), 787 - 9 Two contrary modes of chemolithotrophy in the same archaebacterium; Segerer A et al.; Sulphur-dependent archaebacteria, which are found around nearly boiling continental solfataric springs and mud holes, can be assigned to two distinct branches: the aerobic, sulphur-oxidizing Sulfolobales and the strictly anaerobic sulphur-reducing Thermoproteales . Here, we report the isolation of a group of extremely thermophilic solfataric archaebacteria that are able to grow either strictly anaerobically by reduction, or fully aerobically by oxidation of molecular sulphur, depending on the oxygen supply . We have also established that the ability to grow in these two ways is shared by Sulfolobus brierleyi, a well-known less thermophilic sulphur-oxidizing archaebacterium capable of ore-leaching . The phenomenon may be dependent on a fundamental switch in genome expression . These organisms might represent the primitive fore-runners of sulphur-oxidizing archaebacteria, meeting their energy requirements either by oxidation or by reduction of the same element. J Protozool, 1985 Feb, 32(1), 32 - 7 S-Adenosylhomocysteine hydrolase deficiency in cysteine auxotrophs of Tetrahymena thermophila; Murphy SE et al.; The biochemical lesion in two cysteine auxotrophs of Tetrahymena thermophila has been established as a defect in S-adenosylhomocysteine hydrolase, an enzyme of the transsulfuration pathway . As a result, these mutants require cysteine (or cystathionine or homocysteine) for growth in a defined medium . Cell-free extracts of the mutants contained less than 5% of the level of the enzyme seen in the wild type . One of the mutant strains accumulated intracellular levels of S-adenosylhomocysteine as high as 1380 microM, a level 200 times normal . When both mutant strains were maintained in defined medium without cysteine, growth occurred after a long lag; this phenomenon was termed "adaptation." Adaptation was a) reversed by passage through rich medium, b) was not a recovery of S-adenosylhomocysteine hydrolase, and c) was probably linked to induction of an alternate pathway for cysteine biosynthesis, involving a lysosomal S-adenosylhomocysteine nucleosidase activity. J Protozool, 1985 Feb, 32(1), 126 - 39 Oral ultrastructure and oral development of the misaligned undulating membrane mutant of Tetrahymena thermophila; Lansing TJ et al.; The misaligned undulating membrane (mum) mutant of Tetrahymena thermophila is a non-conditional, single gene recessive mutation . The major effect of the mum mutation is the production of multiple undulating membrane (UM) fragments in the oral apparatus (OA) . The ultrastructure of the UM fragments of mum OAs is identical to that of the single UM of wild-type OAs . Analysis of OA development at midbody using a combination of light microscopy of protargol-stained cells and SEM of demembranated whole cells showed that the phenotypic effect of the mum mutation first becomes evident during mid to late stage 4 and is fully manifested in early stage 5 . The effect of the mutation involves a proliferation of excess basal bodies in the UM field . Subsequent events in the development of the mum OA from mid to late stage 5 are identical to those in wild-type OAs . This study suggests that the mum mutation establishes conditions that allow the production of multiple UMs and thus reveals that the UM field is competent for the complete and coordinated development of several adjacent UMs . This level of regional control is not clearly evident when a single UM is present . The comparison of development of wild-type and mum OAs required an extensive reanalysis of stages 4 and 5 of normal oral development . On the basis of current and previous observations, we propose a new and more subdivided staging system for oral development in Tetrahymena. J Dairy Res, 1985 Feb, 52(1), 197 - 207 Nutritional value of yogurt; Hewitt D et al.; Yogurt, made from fortified skim milk by conventional methods using Streptococcus thermophilus and Lactobacillus bulgaricus, was used in studies of the effect of fermentation on nutritional value of milk . In all experiments, the product was compared with the uninoculated base milk . The concentration of most vitamins was less in yogurt than in milk and was most noticeably so for biotin which was 60% less . The effect on folic acid content was inconsistent . In nutritional experiments with rats, high values for true digestibility, biological value and net protein utilization were obtained for both yogurt and its base milk, only minor differences being apparent between the two materials . In growth tests with rats, yogurt was not found to be consistently superior to the base milk when the milk was subjected to a double heat treatment to reduce bacterial contamination . Yogurt did not confer a nutritional advantage on fresh milk in this respect. J Clin Microbiol, 1985 Feb, 21(2), 222 - 5 Illness associated with Campylobacter laridis, a newly recognized Campylobacter species; Tauxe RV et al.; Campylobacter laridis, a recently described thermophilic Campylobacter species found principally in seagulls, has not previously been linked to illness in humans . Six clinical isolates of this species were referred to the national campylobacter reference laboratory in 1982 and 1983 . Each isolate was confirmed by biochemical characterization and by DNA relatedness studies . The six isolates were obtained during an illness: enteritis in four, severe crampy abdominal pain in one, and terminal bacteremia in an immunocompromised host in one . The infections occurred in persons 8 months to 71 years old . Neither the geographic distribution nor the reports of the patients suggest that seagulls played a direct role in the epidemiology of these infections . This potential human enteric pathogen appears to be clinically, epidemiologically, and microbiologically similar to Campylobacter jejuni and may be mistaken for it if nalidixic acid susceptibility screening is not routinely performed. J Infect Dis, 1985 Feb, 151(2), 227 - 35 Susceptibility of Campylobacter isolates to the bactericidal activity of human serum; Blaser MJ et al.; Although Campylobacter jejuni and related thermophilic organisms are more common human pathogens than are Campylobacter fetus, most bloodstream or systemic isolates are C . fetus . To understand the pathophysiology related to this observation, the authors studied susceptibility to the bactericidal activity of normal human serum of Campylobacter coli, C . jejuni, and C . fetus isolates from feces and blood . In standardized assays, 10 of 15 C . jejuni and related isolates showed 90% kill (mean, 90.6% +/- 5.9); under more stringent conditions, the relatively resistant strains were completely killed . In contrast, all C . fetus strains were highly serum resistant under both standard and stringent conditions . Killing of C . jejuni was ablated by heating serum to 56 C but restored by addition of complement . Both classical and alternative complement pathways may contribute to killing, and adsorption studies demonstrated antibody dependence . Serum resistance may permit systemic infection by C . fetus, whereas complement- and antibody-mediated serum sensitivity of C . jejuni may account for the relative infrequency of systemic invasion. Cell, 1985 Feb, 40(2), 371 - 80 The Tetrahymena rRNA intron self-splices in E . coli: in vivo evidence for the importance of key base-paired regions of RNA for RNA enzyme function; Waring RB et al.; We have developed an in vivo RNA splicing assay for the self-splicing rRNA intron of Tetrahymena thermophila using E . coli as the host . A DNA fragment containing the intron sequence has been cloned into M13mp83 so that expression of the beta-galactosidase alpha-fragment is dependent upon intron excision from the mRNA precursor . Plaque phenotypes correlate well with levels of excised intron RNA . Point mutations were made by oligonucleotide-directed mutagenesis in conserved sequences P, Q, and S . All showed reduced splicing, agreeing with mitochondrial genetic data for S and providing the first direct evidence that P and Q are functionally important . The results support the hypothesis that base-pairing of R with S and P with Q is important for intron structure and function. J Protozool, 1985 Feb, 32(1), 6 - 9 Processing of digestive vacuoles in Tetrahymena and the effects of dichloroisoproterenol; Fok AK et al.; The digestive-lysosomal system in Tetrahymena has been extensively studied; however, the various vacuole stages and the existence of a required processing period prior to defecation have not been clearly defined . In this study the presence of such a required processing period and the rate of DV defecation in Tetrahymena thermophila were determined . Like the cycle in Paramecium, a digestive cycle in Tetrahymena consisted of two periods: the processing period was 45 min and the defecation period was approximately 2 h, making the complete cycle approximately 3 h . During the defecation period vacuole egestion followed the kinetics of a first-order rate reaction and had a rate constant of 0.0187/min and a t1/2 of 37 min (82 min into the cycle) . Using the naphthol AS-TR phosphate-hexazotized rosanilin method to visualize acid phosphatase activity at the light microscopic level, DVs became positive beginning at 10 min . The number of positive DVs increased to a maximum of 13% when DVs were 20-min old and declined to 5-7% beyond 30 min . Although dichloroisoproterenol (DCI) has been reported by others to stimulate vacuole defecation, we found it inhibited the defecation rate . The extent of inhibition depended on the age of the DVs when exposed to DCI . Vacuole formation was completely blocked in cells preexposed to 40 microM DCI for only 10 min; however, upon further exposure, cells could recover from this inhibition . The time required for complete recovery increased with increasing DCI concentrations . If DCI was given to cells simultaneously with latex beads, it was found to exert a dose-dependent inhibitory effect on DV formation.(ABSTRACT TRUNCATED AT 250 WORDS) Arch Biochem Biophys, 1985 Feb 1, 236(2), 567 - 75 The varied responses of different F1-ATPases to chlorpromazine; Bullough DA et al.; The effects of chlorpromazine on various properties of the F1-ATPases from bovine heart mitochondria (MF1), the plasma membranes of Escherichia coli (EF1), and plasma membranes of the thermophilic bacterium PS3 (TF1) have been examined . While chlorpromazine inhibited MF1 with an I0.5 of about 50 microM and EF1 with an I0.5 of about 150 microM at 23 degrees C, the ATPase activity of TF1 was stimulated by chlorpromazine concentrations up to 0.6 mM at this temperature . Maximal activation of about 20% was observed at 0.2 mM chlorpromazine at 23 degrees C . Chlorpromazine concentrations greater than 0.6 mM inhibited TF1 at 23 degrees C . At 37 degrees C the ATPase activity of TF1 was doubled in the presence of 0.5 mM chlorpromazine, the concentration at which maximal stimulation was observed at this temperature . Chlorpromazine inhibited the rate of inactivation of EF1 by dicyclohexylcarbodiimide (DCCD) at 23 degrees C and pH 6.5 . Concentrations of chlorpromazine which inhibited the ATPase activity of TF1 at pH 7.0 accelerated the rate of inactivation of the enzyme by DCCD at pH 6.5, while lower concentrations of the phenothiazine, which stimulated the ATPase, had no effect on DCCD inactivation . Chlorpromazine concentrations up to 1.0 mM had no effect on the rate of inactivation of TF1 by DCCD at 37 degrees C and pH 6.5 . Chlorpromazine at 0.5 mM accelerated the rate of inactivation of MF1 by 5'-p-fluorosulfonylbenzoyladenosine (FSBA), while it slowed the rate of inactivation of EF1 by FSBA . The inactivation of TF1 by FSBA in the absence of chlorpromazine was complex and was not included in this comparison . Chlorpromazine protected MF1 and EF1 against cold inactivation . Whereas 100 microM chlorpromazine afforded about 90% stabilization of MF1 at 4 degrees C, only about 30% stabilization of EF1 was observed under the same conditions in the presence of 400 microM chlorpromazine . Each of the ATPases was inactivated by the structural analog of chlorpromazine, quinacrine mustard . Whereas 5 mM ATP and 5 mM ADP protected MF1 and TF1 against inactivation by 0.5 mM quinacrine mustard, the rate of inactivation of EF1 by quinacrine mustard was accelerated fourfold by 5 mM ATP and slightly accelerated by 5 mM ADP. Nucleic Acids Res, 1985 Jan 11, 13(1), 73 - 87 DNA synthesis, methylation and degradation during conjugation in Tetrahymena thermophila; Harrison GS et al.; We have investigated the timing of DNA synthesis, methylation and degradation during macronuclear development in the ciliate, Tetrahymena thermophila . DNA synthesis was first detected in the anlagen early in macronuclear development, but the majority of DNA synthesis occurred later, after pair separation . Anlagen DNA was first detectably methylated at GATC sites 3-5 hours after its synthesis . Once initiated, de novo methylation was rapid and complete, occurring between 13.5 and 15 hours of conjugation . The level of methylation of GATC sites was constant throughout the remainder of conjugation, and was similar to that in mock-conjugated cells . Degradation of DNA in the old macronucleus and DNA synthesis in the anlagen began at about the same time . Upon pair separation, less than 20% of old macronuclear DNA remained . A small percentage of nucleotides prelabeled prior to conjugation were recycled in the developing anlagen. Nucleic Acids Symp Ser, 1985, (16), 225 - 8 Two types of tRNA(Gm)methylase found in extreme thermophile, Thermus thermophilus strains HB 8 and