Thermophile

J Biol Chem, 1983 Jul 25, 258(14), 8543 - 6
Mössbauer study of beef heart cytochrome oxidase . Comparative study of the bovine enzyme and cytochrome c1aa3 from Thermus thermophilus; Kent TA et al.; We have studied beef heart cytochrome c oxidase at 4.2 K with Mossbauer spectroscopy using the 57Fe present in natural abundance . The spectra observed are very similar to those of the a- and a3-sites of cytochrome c1aa3 from Thermus thermophilus . Thus, many conclusions derived from studies of the bacterial oxidase (available with enriched 57Fe) also apply to the mammalian enzyme . In the resting (as isolated) state, cytochrome a3 of the mammalian enzyme exhibits a doublet with quadrupole splitting, delta EQ = 1.0 mm/s and isomer shift, delta = 0.48 mm/s . These parameters suggest a high spin ferric heme and rule out an Fe(IV) assignment . The absence of magnetic features in the 4.2 K spectrum is consistent with earlier proposals that cytochrome a3 is spin-coupled to a cupric ion . The absorption lines are rather broad, suggesting that the a3-site is heterogeneous in the resting enzyme . Reduced cytochrome a3 has delta EQ = 1.85 mm/s and delta = 0.93 mm/s, demonstrating that the heme iron is high spin ferrous . The observed value for delta EQ is smaller than those of hemoglobin (2.4 mm/s), myoglobin (2.2 mm/s), and cytochrome a3 from T . thermophilus (2.06 mm/s) . The Mossbauer spectra of oxidized cytochrome a3-CN show that the heme iron is low spin ferric and that the ground state has integer spin S greater than or equal to 1, which plausibly results from ferromagnetic coupling of the S = 1/2 heme to an S = 1/2 cupric ion . Reduced cytochrome a is low spin ferrous, with parameters similar to those of cytochrome b5 and cytochrome c.

Anal Biochem, 1983 Jul 15, 132(2), 413 - 7
The application of triazine dye affinity chromatography to the large-scale purification of glycerokinase from Bacillus stearothermophilus; Scawen MD et al.; Gram quantities of homogeneous glycerokinase have been prepared from the thermophilic bacterium, Bacillus stearothermophilus, using three major steps: precipitation of debris at pH 5.1, ion-exchange chromatography on DEAE-Sephadex, and affinity chromatography on Procion Blue MX-3G-Sepharose . This method is a considerable improvement over conventional techniques; the purified enzyme was obtained with a 40% recovery and a specific activity of 120 units (mumol/min)/mg protein . A modified culture medium enabled yields of 3.4 X 10(6) units of enzyme to be obtained from 400-liter production cultures.

J Bacteriol, 1983 Jul, 155(1), 90 - 6
Temperature dependence of growth and membrane-bound activities of Chloroflexus aurantiacus energy metabolism; Oelze J et al.; The temperature dependence of various activities related to the energy metabolism of isolated membranes and whole cells of the thermophilic bacterium Chloroflexus aurantiacus was determined after phototrophic growth at either 40, 50, or 60 degrees C . The data obtained were expressed by use of Arrhenius plots . Maximum activities were determined at about 65 degrees C for succinate 2,4-dichlorophenol-indophenol reductase as well as NADH oxidase and at about 70 degrees C for Mg-ATPase and for light-induced proton extrusion by cells . Activation energies for Mg-ATPase and light-induced proton extrusion were about 40 kJ mol-1 from 30 degrees C to about 50 degrees C and they increased significantly at higher temperatures . Essentially the same dependency was detectable with NADH oxidase, except for an increase in activation energy below 41 degrees C . All of these responses were independent of growth temperature . Succinate-2,4-dichlorophenol-indophenol reductase showed a change in activation energy around 41 degrees C only with cells grown at 60 degrees C . Differences in the responses of cells grown at different temperatures were identified on the basis of changes from sigmoidal to hyperbolic kinetics for light saturation of proton extrusion . Moreover, the thermostability of proton extrusion was maximal when assayed at the corresponding growth temperatures . In any case, thermostability was lowest at the 65 and 68 degrees C assay temperatures . Differential scanning calorimetry with membranes revealed irreversible heat uptake from about 60 to 72 degrees C . The results are discussed in light of the activation energy for the specific growth rate, which is lowest at temperatures from 40 degrees C to the optimum at 60 degrees C.

Dev Biol, 1983 Jul, 98(1), 173 - 81
An analysis of protein synthesis, membrane proteins, and concanavalin A-binding proteins during conjugation in Tetrahymena thermophila; Van Bell CT; Conjugation in the ciliate Tetrahymena thermophila has been used as a system in which to analyze biochemical events associated with the execution of a complex cell-cell interaction . Two-dimensional electrophoretic analysis of {35S}methionine-labeled whole-cell proteins revealed major changes in protein synthesis correlated with costimulation and the onset of pairing; specifically, the major induced polypeptide was one of 80 kDa . A second change in the pattern of protein synthesis was associated with the onset of meiosis; the major induced product was another, perhaps related, 80-kDa polypeptide . An effort was made to detect changes in the patterns of membrane proteins and Con A-binding proteins during conjugation; no changes were found . These results are discussed in the context of earlier hypotheses regarding the distribution of Con A receptors on the surfaces of conjugating cells.

Mikrobiologiia, 1983 Jul-Aug, 52(4), 605 - 8
{Thermophilic Myceliophthora thermophila decomposes cellulose}; Loginova LG et al.; A thermophilic microscopic fungus was isolated from cattle rumen and identified as Myceliophthora thermophila (Apinis) van Oorschot . The culture synthesized cellulolytic enzymes and xylanase when it was grown in media containing cellulase at 50 degrees C under the conditions of submerged cultivation . The morphological and physiological characteristics of the culture are described and its taxonomic position is discussed.

Res Commun Chem Pathol Pharmacol, 1983 Jul, 41(1), 149 - 55
Effect of various compounds on enkephalin hydrolysis by an aminopeptidase from the thermophiles Thermomonospora fusca ATCC 27730 and Thermus thermophilus ATCC 27634; Weiss B et al.; The microbial peptides amastatin and bestatin as well as several dipeptide analogues of the latter exerted little or no inhibitory effect on enkephalin hydrolysis by an aminopeptidase purified from the thermophiles Thermomonospora fusca, ATCC 27730 (Tf) and Thermus thermophilus, ATCC 27634 (Tt) . The enzyme catalyzes the cleavage of the tyrosyl-glycyl bond of leucine- and methionine-enkephalin . Intermediate compounds having the same amino acid sequence as the parent substrate disclosed that the residual tetrapeptide can be further degraded to its constituent parts . Each preparation also hydrolyzes to varying extents neutral dipeptides, tripeptides, tetrapeptides, can be further degraded to its constituent parts . Each preparation also hydrolyzes to varying extents neutral dipeptides, tripeptides, tetrapeptides, and larger molecules containing the Met-enkephalin sequence . The Tf enzyme has a pH optimum of 7.5, Km of 667 microM and Vmax of 92 nmol/min/mg of protein; the Tt enzyme, with a pH optimum of 7.2 has a Km of 400 microM and Vmax of 33 nmol/min/mg of protein . Activated by dithiothreitol (DTT) and inactivated by p-chloro- and p-hydroxymercuribenzoate, both are sulfhydryl enzymes . The activity lost by hydrolysis against EDTA can be restored, wholly or in part, by Co+2, Mg+2, and Mn+2; ions with an inhibitory effect were A1+3, Cd+2, Cu+2, Hg+2, and Zn+2 . The enzymes are not glycoproteins since they pass unretained through a Con A-Sepharose column.

Hoppe Seylers Z Physiol Chem, 1983 Jul, 364(7), 879 - 92
Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria . II) The primary structure of thermophilic lactate dehydrogenase from Bacillus stearothermophilus . Cyanogen bromide fragments and partial sequence; Tratschin JD et al.; The polypeptide chain of thermophilic lactate dehydrogenase from Bacillus stearothermophilus was split with cyanogen bromide . The 6 cyanogen bromide fragments were then separated and isolated by gel filtration (Bio-Gel P 10, Sephadex G-75) and ionic exchange chromatography (Biorex 70), respectively . Peptide fractionation was performed in 50% formic acid . Fragment yield varied between 30 and 75% . About 75% of the amino-acid sequence was determined by the automatic N-terminal sequence analysis (amino-acid sequenator) of the cyanogen bromide fragments (41-57 cycles degraded) and N-terminal region of lactate dehydrogenase (74 cycles degraded) . Typical structure differences between thermophilic and mesophilic lactate dehydrogenases are already indicated by the comparison of the amino-acid composition of the thermophilic enzyme from B . stearothermophilus with the mesophilic from bacilli and higher organisms . Comparison of the N-terminal sequence reveals that sequence homology is higher (83-98%) between the thermophilic lactate dehydrogenases from B . stearothermophilus, B . caldotenax and B . caldolyticus than between the mesophilic lactate dehydrogenases of bacilli among each other or between thermophilic and mesophilic lactate dehydrogenases (about 60%) . High temperature would appear to limit variation in structure.

Radiobiologiia, 1983 Jul-Aug, 23(4), 462 - 6
{Effect of MEA on the accumulation of DNA breaks in Bac . stearothermophilus irradiated with gamma and ultraviolet rays and treated with nitrosomethylurea}; Kuznetsova EA et al.; beta-Mercaptoethylamine (MEA) decreased the accumulation of enzymatic single- and double-strand breaks in DNA of thermophilic bacteria exposed to gamma- and UV-radiation and treated with N-nitroso-N-methylurea . The protective effect of MEA, as registered according to accumulation of single-strand and double-strand breaks in DNA of Bac . stearothermophilus immediately after irradiation and after 30 min postirradiation incubation, was similar.

Equine Vet J, 1983 Jul, 15(3), 207 - 10
Chronic obstructive pulmonary disease in the horse . 2: Therapy; Thomson JR et al.; The therapy of equine chronic obstructive pulmonary disease (COPD) essentially entails minimising the horse's exposure to the aetiological antigens which are predominantly thermophilic actinomycetes and moulds occurring in hay and straw . This can be achieved, for example, by keeping affected horses permanently out of doors, or when stabled, using shredded paper, wood shavings or peat moss as bedding and feeding a complete cubed diet . There should be no supplementary hay feeding apart from dust-free vacuum-packed hay . Applying such measures generally allows horses to become asymptomatic in seven to 14 days . Bronchodilators and corticosteroids bring about a marked, but temporary, improvement and can be of value in the treatment of acute attacks . The use of oral bronchodilators in combination with environmental control measures may hasten the remission of clinical signs in affected horse . Inhaled sodium cromoglycate can be used prophylactically in asymptomatic horses to prevent the onset of COPD when unavoidable antigen exposure is anticipated.

J Clin Pathol, 1983 Jul, 36(7), 829 - 34
A study of the oxygen and carbon dioxide requirements of thermophilic campylobacters; Bolton FJ et al.; The oxygen and carbon dioxide requirements of different biotypes of thermophilic campylobacters were investigated by means of (a) quantitative studies, and (b) total growth studies . Oxygen tolerance of the five test organisms differed markedly and varied with the carbon dioxide concentration . At most carbon dioxide concentrations tested, Campylobacter jejuni strains NCTC 11168 and NCTC 11392 tolerated 21% oxygen (growth reduced), C coli NCTC 11353 tolerated 15% oxygen (growth reduced), and C jejuni ATCC 3036 and (nalidixic acid resistant thermophilic campylobacter) NCTC 11352 tolerated 10% oxygen (growth not reduced) . Total growth studies indicated that 10% oxygen was the optimal concentration for growth of the five test organisms . All exhibited a requirement for carbon dioxide, and only C jejuni strains NCTC 11168 and NCTC 11392 tolerated its absence (growth reduced), when the oxygen concentration was low . The studies indicated that atmospheres containing 5% to 10% oxygen and 1.0% to 10% carbon dioxide are suitable for growth of the various biotypes of thermophilic campylobacters . The oxygen and carbon dioxide concentrations produced in anaerobic jars by variations of the evacuation-replacement technique were determined and suitable practices identified.

Hoppe Seylers Z Physiol Chem, 1983 Jul, 364(7), 893 - 909
Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria . III) The primary structure of thermophilic lactate dehydrogenase from Bacillus stearothermophilus . Hydroxylamine-, o-iodosobenzoic acid- and tryptic-fragments . The complete amino-acid sequence; Wirz B et al.; Based on the partial sequence of the cyanogen bromide fragments {Tratschin, J.D., Wirz, B., Frank, G . and Zuber, H . (1983) Hoppe-Seyler's Z . Physiol . Chem . 364, 879-892}, the amino-acid sequence of thermophilic lactate dehydrogenase from B . stearothermophilus was completed by the preparation and sequencing (sequenator, carboxypeptidase A and Y) of further overlapping fragments . Suitable peptide fragments were obtained by lactate dehydrogenase cleavage with hydroxylamine, o-iodosobenzoic acid and trypsin . The polypeptide chain of thermophilic lactate dehydrogenase from B . stearothermophilus consists of 317 amino-acid residues . While sequence homology with mesophilic lactate dehydrogenase of higher organisms reaches 35%, it is substantially higher with this mesophilic enzyme of bacillae (greater than 60%, B . megaterium, B . subtilis) . The secondary structure elements and amino-acid residues of the active site of thermophilic lactate dehydrogenase deducted from primary structure data were compared with those from the mesophilic enzyme, the same was done for the internal sequence homology at the nucleotide-binding units . A comparative structure analysis (matrix system) based on the primary structure data of thermophilic enzyme should provide insight into the characteristic structure differences between thermophilic and mesophilic lactate dehydrogenase.

FEBS Lett, 1983 Jun 27, 157(1), 95 - 9
NMR analyses on the molecular mechanism of the conformational rigidity of 2-thioribothymidine, a modified nucleoside in extreme thermophile tRNAs; Yamamoto Y et al.; 1H-NMR analyses have been made on the conformations of 2-thioribothymidine (s2T), 2-thiodeoxyribothymidine (s2dT), as well as ribothymidine (T) and deoxyribothymidine (dT) . s2T and s2dT exclusively take the anti form rather than the syn form . The C3'-endo-gg form of the sugar moiety is remarkably stabilized on modification of T to s2T, but not on modification of dT to s2dT . The steric effects of the 2-thiocarbonyl group and the 2'-hydroxyl group cause the rigidity of the C3'-endo-gg form of s2T . Such rigidity of s2T probably contributes to the thermostability of 2-thiopyrimidine polyribonucleotides and extreme thermophile tRNAs.

Biochem Biophys Res Commun, 1983 Jun 15, 113(2), 575 - 80
Proton pumping and oxidase activity of thermophilic cytochrome oxidase remain after its extensive proteolysis; Yanagita Y et al.; A proton-pumping heme aa3-type cytochrome oxidase purified from the thermophilic bacterium PS3 was treated with trypsin, thermolysin, chymotrypsin, subtilisin, or pronase . The cleavage of the oxidase subunits and the effects of their cleavage on the oxidase activity and proton-pumping in reconstituted vesicles were studied . Trypsin and thermolysin cleaved some of the oxidase subunits without affecting the proton-pumping, but subtilisin and pronase cleaved all the subunits resulting in partial decrease in both activities . Chymotrypsin had an intermediate effect . Subunit II of this enzyme contains heme c which is also cleaved by proteases.

Nucleic Acids Res, 1983 Jun 11, 11(11), 3487 - 502
Complex endonucleolytic cleavage pattern during early events in the processing of pre-rRNA in the lower eukaryote, Tetrahymena thermophila; Kister KP et al.; We have analysed nuclear RNA from T . thermophila by RNA transfer hybridization using cloned rDNA fragments . A very high number of in vivo intermediates and by-products of rRNA processing were identified . These include putative intermediates of the splicing process and alternative products resulting from temporal variability in various endonucleolytic cleavages . In addition, four small RNA species including only transcribed spacer sequences were detected . These are (1) the IVS RNA (approximately 400 bases), the by-product of the splicing process, (2) a fragment from the internal transcribed spacer (approximately 360 bases), possibly resulting from 3'-end processing of pre-17S rRNA, (3) a fragment comprising most or all of the external transcribed spacer (approximately 600 bases) obviously representing the major by-product of 5'-end processing, and, in addition, (4) a small fragment from the initiation region (approximately 230 bases) which might be a product of premature transcription termination.

J Biol Chem, 1983 Jun 10, 258(11), 6899 - 905
Regulation of protein synthesis in Tetrahymena . RNA sequence sets of growing and starved cells; Calzone FJ et al.; The complexity of messenger RNA in growing or starved Tetrahymena thermophila is similar and unusually high (approximately 4.5 X 10(7) nucleotides) . The complexity of nuclear RNA in growing cells (approximately 7.8 X 10(7) nucleotides) is only about 1.7 times that of mRNA . The concentration of complex class (rare) messages (approximately 53 copies/growing cell and approximately 11 copies/starved cell) is low in comparison to the size of the cell . The concentration of complex nuclear transcripts is also very low (approximately 0.7 copies/growing cell nucleus and approximately 2.6 copies/starved cell nucleus) considering that the macronucleus contains 45 to 90 copies of each single copy sequence . The complex sequence sets found on polysomes of growing and starved cells overlap about 80% and about 60% of the complex nuclear transcripts appear to be held in common . About 60% of macronuclear single copy DNA is transcribed in one or both physiological states . Although growing and starved cells have extremely different fractions of their messages loaded onto polysomes, within each cell type the complex messages in polysomal and nonpolysomal cytoplasmic fractions are indistinguishable, suggesting that exchange may occur between loaded and unloaded messages . Although T . thermophila DNA has an unusually low G + C content (23%), sequences coding for complex RNAs have base ratios similar to those of total DNA . Therefore, codon usage in Tetrahymena must be extremely biased towards adenine- and uridine-rich codons.

Arch Microbiol, 1983 Jun, 134(3), 247 - 50
Changes in enzyme stability and fatty acid composition of Streptomyces sp., a facultative thermophilic actinomycete; Heinen W et al.; The thermostability of several enzymes from the facultative thermophilic actinomycete Streptomyces sp., derived from cells grown in the temperature range from 37 degrees C to 60 degrees C, has been examined . A correlation between the growth temperature of the cultures and the heat stability of the enzymes could be demonstrated for alanine dehydrogenase, isocitrate dehydrogenase, myokinase and pyruvate kinase . Except for the isocitrate dehydrogenase, which showed a linear increase of its stability throughout the entire temperature range, all enzymes exhibited a steep increase of the heat stability up to about 50 degrees C, but no further increase in the higher growth range, suggesting, that from 50 degrees C upward a shift to the exclusive production of thermostable protein occurs . Furthermore, the stability of alanine dehydrogenase and pyruvate kinase was found to increase substantially in presence of their substrates . In contrast, substrate-mediated stabilization was very weak with glucose-6-phosphate dehydrogenase and totally absent with acetate kinase, isocitrate dehydrogenase and myokinase . A comparison with previous observations with the same enzymes from Bacillus flavothermus showed, that enzymes from different organisms can have different thermal properties . Determination of the fatty acid composition of Streptomyces sp . cells, grown at different temperatures, showed relatively small alterations, with the main changes occurring between 37 degrees C and 40 degrees C.

Arch Microbiol, 1983 Jun, 134(3), 175 - 81
Activation and germination characteristics observed in endospores of thermophilic strains of Bacillus; Foerster HF; The properties of endospores of some thermophilic strains of Bacillus were examined . Included were strains isolated from thermal pools and springs in Yellowstone National Park, a strain of B . thermodenitrificans and two strains of B . stearothermophilus, ATCC 7953 (smooth) and T-10 . The spores of thermophilic strains of Bacillus contained relatively high levels of dipicolinic acid ranging from 11-14.8% of the spore dry weight, while the calcium levels were similar to those observed in other bacterial endospores including mesophilic bacilli and thermophilic actinomycetes . Spore populations of thermophilic bacilli could not be effectively germinated in solutions of sodium phosphate alone but germinated well in solutions supplemented with one of a variety of organic compounds . Solutions containing L-valine or L-leucine were particularly effective . A wide range of pH permitted the germination of fractions of spore populations, however, optimum germination was observed only at pH values of 6.0 and above . A range in incubation temperatures of less than 25 degrees C permitted 50% or more of the spores of each of the organisms to germinate . Freshly prepared spore did not germinate, but these spores germinated rapidly and completely if they were heated for 30 min at 100 degrees C just prior to germination testing, i.e., the spores were heat activatable . However, spores of thermophilic bacilli could also be activated by shifting them to and holding them at temperatures below their optimum growth temperature of 65 degrees C . Of the ten temperatures tested, ranging from 4 degrees C through 50 degrees C, the optimum reduced temperature for spore activation was 30 degrees C.

Hoppe Seylers Z Physiol Chem, 1983 Jun, 364(6), 691 - 712
The complete amino-acid sequence of both subunits of phycoerythrocyanin from the thermophilic cyanobacterium Mastigocladus laminosus; Fuglistaller P et al.; The amino-acid sequences of both subunits of phycoerythrocyanin from the thermophilic cyanobacterium Mastigocladus laminosus have been determined . The alpha-subunit consists of 162 amino-acid residues and has a molecular mass of 18200 Da . The beta-subunit is 171 residues long and has a molecular mass of 19600 Da . The tetrapyrrole chromophores are bound at position 84 in the alpha- and beta-subunits and at position 155 in the beta-subunit . The homology between the two subunits is 21% . The homologies between the phycoerythrocyanin subunits and the corresponding subunits of C-phycocyanin and allophycocyanin are 63% and 26% for the alpha-subunits and 67% and 36% for the beta-subunits, respectively . Secondary structure predictions were calculated for all six subunits of the phycobiliproteins from M . laminosus . The most conservative regions of the biliproteins were found in segments C-terminal to the chromophore-binding sites.

Proc Natl Acad Sci U S A, 1983 Jun, 80(11), 3406 - 10
Mitotic and meiotic stability of linear plasmids in yeast; Dani GM et al.; Circular recombinant DNA plasmids that contain autonomously replicating sequences (ARSs) are maintained in extrachromosomal form in transformed yeast cells . However, these plasmids are unstable, being rapidly lost from cells growing without selection . Although the stability of such a plasmid can be increased by the presence of yeast centromere DNA (CEN), even CEN plasmids are lost at a high rate compared to a bona fide yeast chromosome . Natural yeast chromosomes are linear molecules; therefore, we have asked if linearization can improve the stability of recombinant DNA plasmids . Linear plasmids with and without yeast CENs were constructed in vitro by using termini from the extrachromosomal ribosomal DNA (rDNA) of the ciliated protozoan Tetrahymena thermophila as "telomeres." These linear plasmids transformed yeast at high frequency and were maintained as linear extrachromosomal molecules during mitotic growth . Moreover, linear plasmids containing CENs were also transmitted through meiosis: these plasmids segregate predominantly 2+:2- at the first meiotic division, indicating that Tetrahymena rDNA termini can provide telomere function during yeast meiosis . Linear plasmids without CENs were about as stable in mitosis as the comparable circular plasmid . Thus, the Tetrahymena rDNA termini have no marked positive or negative effect on the mitotic stability of ARS plasmids . However, linear plasmids containing CENs are three to four times less stable in mitotic cells than circular CEN plasmids . This decrease in stability is not due to a functional change in the centromere itself; rather, linearization of a CEN plasmid has a direct detrimental effect on its mitotic stability . These results may reflect the existence of spatial constraints on the positions of centromeres and telomeres, constraints which must be satisfied to achieve stable segregation of chromosomes during mitosis.

Antonie Van Leeuwenhoek, 1983 Jun, 49(2), 191 - 201
Thermal properties of enzymes from Bacillus flavothermus, grown between 34 and 70 degrees C; Lauwers AM et al.; The activity and stability of several enzymes from the facultative thermophile Bacillus flavothermus, grown within the mesophilic and thermophilic region at 34 degrees C, 43 degrees C, 52 degrees C and 70 degrees C, have been examined . While the temperature optima and maxima of all enzymes tested were found to remain unchanged at all growth temperatures, it was demonstrated that the heat stability of the proteins increased with ten perature, however, not uniformly for all enzymes . One exception was acetate kinase and the intrinsic stability of pyruvate kinase was found to increase only slightly . With all other proteins tested (alanine dehydrogenase, isocitric dehydrogenase and glucose-6-phosphate dehydrogenase, glutamate-oxalacetate and glutamate-pyruvate transaminase and myokinase) the intrinsic stability was found to increase to about 55 degrees C, but stayed unaltered at higher growth temperatures . Except for acetate kinase and myokinase, the enzymes could be stabilized by their respective substrates and the heat stability of the ES-complexes was found also to depend on the growth temperature of the cells . These data lead to the conclusion that the enzymes undergo a transition from heat-labile to thermostable within the growth temperature range between 44 degrees C and 51 degrees C while the thermal characteristics are not changed below and beyond this crucial region.

J Bacteriol, 1983 Jun, 154(3), 1451 - 4
Base excision repair in the thermophile Thermus sp . strain X-1; Warner HR; The thermophile Thermus sp . strain X-1, grown at 70 degrees C, contains uracil-DNA glycosylase and apurinic endonuclease activities, both of which are known to have roles in the repair of DNA damaged by heat . Both of these activities have temperature optima of about 70 degrees C . However, neither of these activities is present in quantities significantly greater than that found in Escherichia coli grown at 37 degrees C . Therefore, it appears that thermophilic organisms may not contain greatly elevated levels of the enzymes thought to be involved in the repair of DNA damaged by heat.

Biochem Biophys Res Commun, 1983 May 16, 112(3), 822 - 6
Resistance of thermophilic ATPase (TF1) to specific F1-atpase inhibitors including local anesthetics; Saishu T et al.; F1-ATPase obtained from mesophilic organisms is inhibited by specific inhibitors, such as aurovertin, efrapeptin, quercetin and several local anesthetics . This property has been explained by the common structure at the catalytic center of F1 . However thermophilic F1 (TF1), which has the same primary structure at the center as other F1's, was shown to be resistant to these F1-specific inhibitors . Thus, the inhibitory mechanism may be explained not by the common structure at the catalytic site, but by some conformational changes of the flexible mesophilic F1 molecules or the absence of an inhibitor binding site in thermophilic F1.

Biochemistry, 1983 May 10, 22(10), 2314 - 9
Redox-linked hydrogen bond strength changes in cytochrome a: implications for a cytochrome oxidase proton pump; Babcock GT et al.; The heme a formyl group of cytochrome a in cytochrome oxidase appears to be involved in a hydrogen-bond interaction with a proton donor associated with the polypeptide backbone {Callahan, P.M., & Babcock, G.T . (1983) Biochemistry 22, 452-461} . Resonance Raman and optical absorption spectroscopies have been applied to the beef heart and Thermus thermophilus proteins and to heme a and copper porphyrin a models in order to assess the spectroscopic manifestations and the energetics of the hydrogen-bond interaction . We find a linear relationship between optical absorption red shift and carbonyl vibrational frequency decrease for a series of hydrogen-bonded model complexes; the magnitude of both changes increases as the hydrogen-bond strength increases . Comparison of the model compound data with analogous data for the proteins indicates that the strength of the formyl hydrogen bond in situ increases by 2-2.5 kcal/mol upon reduction of ferric cytochrome a . The selective stabilization of reduced cytochrome a by the stronger hydrogen bond is expected to increase the redox potential of this center; the energy made available as the hydrogen bond strengthens during reduction may be used to drive redox-coupled events in the protein . Thus, the linkage between cytochrome a redox state and chromophore/protein interaction energy provides a mechanism by which electron-transfer events and protein structure are coupled . Two models, which incorporate this linkage into a redox-driven proton pump centered at cytochrome a in cytochrome oxidase, are presented.

FEBS Lett, 1983 May 2, 155(1), 173 - 7
Ribosome specificity of archaebacterial elongation factor 2 . Studies with hybrid polyphenylalanine synthesis systems; Klink F et al.; Polyphenylalanine synthesis with ribosomes and two separated, partially purified elongation factors (EF) was measured in cell-free systems from the archaebacteria Thermoplasma acidophilum and Methanococcus vannielii, in an eukaryotic system from rat liver and an eubacterial one with Escherichia coli ribosomes and factors from Thermus thermophilus . By substitution of heterologous EF-2 or EF-G, respectively, for the homologous factors, ribosome specificity was shown to be restricted to factors from the same kingdom . In contrast, EF-1 from T . thermophilus significantly cooperated with ribosomes from T . acidophilum.

J Biochem (Tokyo), 1983 May, 93(5), 1455 - 6
Homocaldopentamine: a new naturally occurring pentaamine; Oshima T et al.; A new pentaamine was extracted from an extreme thermophile, Thermus thermophilus strain HB8, and its chemical structure was determined to be 1,16-diamino-4,8,12-triazahexadecane (see structure 1) . A trivial name, homocaldopentamine, was proposed for the new naturally occurring polyamine.

J Biochem (Tokyo), 1983 May, 93(5), 1329 - 36
Carbon monoxide-binding cytochromes in the respiratory chain of the thermophilic bacterium PS3 grown with sufficient or limited aeration; Sone N et al.; The effects of aeration on the growth and cytochrome patterns of thermophilic bacterium PS3 were studied; bacteria grown with strong aeration synthesized cytochromes c, b, and aa3, while those grown with low aeration, showing non-exponential growth, synthesized higher amounts of cytochromes c and b including o, and a lower amount of cytochrome a (a3) . The CO-difference spectra indicated that the terminal oxidase was cytochrome aa3 for high aeration conditions and the cytochrome o for low aeration conditions . Cytochrome o can be solubilized by Triton X-100 from the membrane fraction of bacteria grown under oxygen-limited conditions . The carbon monoxide complex of cytochrome o, obtained by exposing this extract to CO, was photolyzed and the subsequent rebinding of CO was analyzed; it followed first order kinetics with a rate constant of around 8 s-1 at 25 degrees C . At liquid nitrogen temperature, CO-rebinding did not occur . The CO-difference spectrum of purified cytochrome oxidase sample from the bacteria grown with strong aeration (Sone, N., et al . (1979) FEBS Lett . 106, 39-42) revealed the presence of a small amount of a cytochrome o-like pigment besides cytochrome aa3 . Analysis of the CO complexes of these chromophores showed rate constants of 29-30 s-1 for cytochrome aa3 and 35-42 s-1 for the o-like pigment, indicating that the cytochrome o-like pigment contaminating the purified cytochrome oxidase preparation was not typical cytochrome o.

Arch Biochem Biophys, 1983 May, 223(1), 193 - 201
Effect of modification of membrane phospholipid composition on the activity of phosphatidylethanolamine N-methyltransferase of Tetrahymena; Smith JD; The activity of phosphatidylethanolamine N-methyltransferase is less than 10% of control levels in microsomes prepared from the ciliate protozoan Tetrahymena thermophila whose phospholipid composition had been altered by being cultured on media containing phosphonic acids . The primary modification obtained is decreased levels of phosphatidylethanolamine (J.D . Smith and D.A . Giegel, Arch . Biochem . Biophys., 206, 420-423 (1981) and 213, 595-601 (1982)) . The enzyme protein is present in these cells at normal levels since addition of the substrate phosphatidylethanolamine to the assay system restores enzyme activity of the lipid-modified microsomes to control levels, while the enzyme from control microsomes is not affected by added phosphatidylethanolamine . The microsomal enzyme is inhibited by the anionic phospholipids cardiolipin, phosphatidylglycerol, and phosphatidylinositol and by lysophosphatidylethanolamine while it is activated only by phosphatidylserine in addition to the substrates phosphatidylethanolamine and phosphatidyldimethylethanolamine . The added phosphatidylethanolamine acts directly as a substrate for the methyltransferase rather than acting by merely stimulating utilization of endogenous lipid since added phosphatidyl{14C}ethanolamine is directly converted to phosphatidylcholine . The results suggest that the technique of phosphonic acid-induced modification of lipid composition will be useful for the study of other membrane-bound enzymes.

J Protozool, 1983 May, 30(2), 403 - 9
The pathogenic amoeboflagellate Naegleria fowleri: environmental isolations, competitors, ecologic interactions, and the flagellate-empty habitat hypothesis; Griffin JL; From several surveys of environmental sites, the virulent human pathogen, Naegleria fowleri, was isolated from a pond in Georgia, a sewage treatment plant in Missouri, and from the Potomac and Anacostia rivers near and in Washington, D.C . Widely scattered, sparse populations seemed only a potential threat to human health at the time of sampling . The data support an estimate that the sites sampled contain 10,000 typical, low temperature, bactivorous amoebae for each heat tolerant amoeba able to grow at 45 degrees C . Heat tolerant competitors were much more common than N . fowleri . Naegleria lovaniensis, which is heat tolerant but nonpathogenic, was isolated from and downstream from an open air thermal pollution temperature gradient . Hot piles of composting sewage sludge yielded no amoeboflagellates, many heat tolerant (45-49 degrees C) amoebae, and one thermophilic (52 degrees C) Acanthamoeba . Features of the methods used include two-stage incubation to increase isolation of sparse organisms and distinction of N . fowleri from almost all other amoebae on agar plates . The flagellate-empty habitat hypothesis postulates a general model in which human intervention and/or natural events remove usual competitors and the ability to transform to a motile flagellate confers an advantage in recolonizing.

Infect Immun, 1983 May, 40(2), 553 - 62
Isolation and possible relevance of Thermoactinomyces candidus proteinases in farmer's lung disease; Roberts RC et al.; The thermophilic actinomycetes are the most common etiological agents causing hypersensitivity pneumonitis . Antigen preparations of these organisms contain proteolytic activity . Further investigation of the proteinases of the thermophilic actinomycetes was undertaken to determine whether this activity may contribute directly to the pathogenesis of hypersensitivity pneumonitis and pulmonary mycotoxicosis . The presence of proteolytic activity in aerosolized dust from moldy silage was demonstrated, and antibodies to two proteolytic enzymes from Thermoactinomyces candidus were found in the blood of farmer's lung patients who had been sensitized to this organism . These two enzymes were isolated from culture filtrate antigen preparations that had been partially characterized with respect to the proteolytic activities and their interaction with human serum proteinase inhibitors . Both proteinases belonged to the serine class of endopeptidases . Neither proteinase was inhibited by alpha 1-proteinase inhibitor or alpha 1-antichymotrypsin . Both proteinases were inhibited by alpha 2-macroglobulin . One of the proteinases had elastase activity . Inhalation of these proteinases apparently does occur, and they may induce an inflammatory response in the lungs since they are not inhibited by the main proteinase inhibitors protecting the lung.

Mycopathologia, 1983 Apr 22, 82(1), 61 - 4
Allergenic fungi and actinomycetes in smoking materials and their health implications; Kurup VP et al.; Street marijuana, commercial cigarettes and pipe tobaccos were studied for the presence of fungi and actinomycetes associated with hypersensitivity pneumonitis . Aspergillus species and thermophilic actinomycetes were isolated from the smoking materials . In addition, Aspergillus fumigatus spores were isolated from marijuana smoke, indicating the potential hazard involved in developing serious disease . Precipitin antibodies against fungi, particularly Aspergillus, showed a higher prevalence in marijuana smokers, whereas only very few cigarette smokers and nonsmokers demonstrated antibodies to fungi . Cigarette smokers and nonsmokers showed more or less similar prevelance of antibodies against thermophilic actinomycetes.

Nucleic Acids Res, 1983 Apr 11, 11(7), 2093 - 109
Different nucleosome spacing in transcribed and non-transcribed regions of the ribosomal RNA gene in Tetrahymena thermophila; Gottschling DE et al.; The chromatin structure of the palindromic macronuclear ribosomal RNA genes of Tetrahymena thermophila was probed with micrococcal nuclease . Independent of the state of transcriptional activity, the transcribed region had a shorter nucleosome repeat (184 +/- 3 base pairs) than the non-transcribed central spacer or bulk chromatin (both 200 base pairs) . The transcribed region displayed an increased sensitivity to micrococcal nuclease in rapidly growing cells, which suggested an altered chromatin structure during transcription . At early stages of nuclease digestion, the central spacer appeared to be in a highly structured nucleosomal array . Based on the differences in nucleosome repeat distance and sensitivity to nuclease, we conclude that quite different chromatin structures are maintained in two adjacent regions of the Tetrahymena ribosomal RNA gene . The DNA of the non-transcribed terminal spacer was found to contain sequences which are highly susceptible to micrococcal nuclease, precluding any conclusions about nucleosome structure in this region.

Appl Environ Microbiol, 1983 Apr, 45(4), 1271 - 6
Adaptation of mesophilic anaerobic sewage fermentor populations to thermophilic temperatures; Chen M; Thermophilic (50 degrees C) and obligately thermophilic (60 degrees C) anaerobic carbohydrate- and protein-digesting and methanogenic bacterial populations were enumerated in a mesophilic (35 degrees C) fermentor anaerobically digesting municipal primary sludge . Of the total bacterial population in the mesophilic fementor, 9% were thermophiles (36 x 10(6)/ml) and 1% were obligate thermophiles (4.5 x 10(6)/ml) . Of these 10%, the percentages of bacteria (thermophiles and obligate thermophiles, respectively) able to use specific substrates were further enumerated as follows: bacteria able to digest albumin, casein, starch, and mono- and disaccharides, 30 and 10%; pectin degraders, 10 and 0.2%; cellulose degraders, 2 and 0.06%; methanogens that grow with H2 and CO2, methanol, and dimethylamine, 9 and 1%; methanogens that grow with formate, 8 and 5%; and methanogens that grow with acetate, 25 and less than 0.8% . Shortly after the temperature was elevated from 35 to 50 or 60 degrees C, the digestion of albumin, casein, starch, and mono- and disaccharides was detected, and methane was produced from H2 and CO2 . Methane produced from acetate was not delayed at 50 degrees C, but was delayed by 29 days at 60 degrees C . Methane produced from formate was delayed by 3 days, from methanol by 7 days, and from dimethylamine by 5 days at 50 and 60 degrees C . A 10- and 20-day acclimation period was required for hydrolysis of pectin and cellulose, respectively, at 50 degrees C . Digestion of pectin required 20 days and cellulose longer than 85 days when the temperature was elevated abruptly from 35 to 60 degrees C . The acclimation period for the digestion of pectin and cellulose at 60 degrees C was shortened to 3 and 15 days, respectively, by seeding with a small amount of a culture acclimated to 50 degrees C . The data suggest that enrichment of cellulolytic, pectinolytic, and acetate-utilizing bacteria is crucial for the digestion of sewage sludge at 60 degrees C.

Mol Cell Biol, 1983 Apr, 3(4), 503 - 10
Characterization of a cycloheximide-resistant Tetrahymena thermophila mutant which also displays altered growth properties; Hallberg RL et al.; A cycloheximide-resistant strain of Tetrahymena thermophila, expressing a mutant chx-B gene (Ares and Bruns, Genetics 90:463-474, 1978), displayed very different temperature-dependent growth characteristics than either wild-type cells or another cycloheximide-resistant strain expressing a different mutant gene . Whereas wild-type cells showed an immediate decline in ribosome translocation rates when shifted from 30 to 38 or 40 degrees C, this mutant strain (X-8) showed no such decline . These results directly correlated with the growth rate differences we found for these cells at these temperatures . By genetic analysis, we showed that the phenotype of cycloheximide resistance cosegregated with the ability to grow rapidly at 40 degrees C . Analyses, both direct and indirect, suggested that a number of functional and structural characteristics of the ribosomes from strain X-8 cells are most likely conformationally different from those of wild-type ribosomes.

J Allergy Clin Immunol, 1983 Apr, 71(4), 389 - 93
Marijuana smoking and fungal sensitization; Kagen SL et al.; The possible role of marijuana (MJ) in inducing sensitization to Aspergillus organisms was studied in 28 MJ smokers by evaluating their clinical status and immune responses to microorganisms isolated from MJ . The spectrum of illnesses included one patient with systemic aspergillosis and seven patients with a history of bronchospasm after the smoking of MJ . Twenty-one smokers were asymptomatic . Fungi were identified in 13 of 14 MJ samples and included Aspergillus fumigatus, A . flavus, A . niger, Mucor, Penicillium, and thermophilic actinomycetes . Precipitins to Aspergillus antigens were found in 13 of 23 smokers and in one of 10 controls, while significant blastogenesis to Aspergillus was demonstrated in only three of 23 MJ smokers . When samples were smoked into an Andersen air sampler, A . fumigatus passed easily through contaminated MJ cigarettes . Thus the use of MJ assumes the risks of both fungal exposure and infection, as well as the possible induction of a variety of immunologic lung disorders.

J Dairy Sci, 1983 Mar, 66(3), 444 - 9
Contribution of Streptococcus thermophilus to growth-stimulating effect of yogurt on rats; Wong NP et al.; The origin of the growth-stimulating factor in yogurt was studied in rats fed liquid or freeze-dried diets of milk, yogurt, milks fermented individually by Streptococcus thermophilus and Lactobacillus bulgaricus, milks to which cells of Streptococcus thermophilus and Lactobacillus bulgaricus were added . Diets containing sonicated cells, cell supernatant, and cell fractions also were fed . Milk fermented by Streptococcus thermophilus and milk plus Streptococcus thermophilus cells stimulated growth as effectively as did yogurt . That finding and the absence of stimulation in rats fed Lactobacillus bulgaricus showed that Streptococcus thermophilus is responsible for stimulation of growth by yogurt . Growth was stimulated by an intracellular factor and not by fermentative changes in the milk.

J Bacteriol, 1983 Mar, 153(3), 1266 - 71
Bacterial elongation factor Ts: isolation and reactivity with elongation factor Tu; Wittinghofer A et al.; An improved method for the purification of bacterial polypeptide elongation factor Ts (EF-Ts) from one mesophile (Escherichia coli) and two thermophiles (Bacillus stearothermophilus and PS3) is described . The improvements are both in the facility of isolation and in increased yields . The purified factors were used for cross-reactivity studies with elongation factor Tu (EF-Tu) obtained from the same bacterial strains . In all combinations studied, the efficiency of EF-Ts in catalyzing the exchange of EF-Tu-bound GDP was proportional to the strength of the protein-protein complex . Whereas the factors from the two thermophiles were interchangeable, the mesophilic EF-Ts formed a very weak complex with thermophilic EF-Tu; however, thermophilic EF-Ts formed very strong complexes with mesophilic EF-Tu . Thus, e.g., EF-Tu from E . coli formed a complex with EF-Ts from B . stearothermophilus which was 10 times more stable than the corresponding homologous complex.

Nature, 1983 Feb 10, 301(5900), 511 - 3
Chemolithoautotrophic metabolism of anaerobic extremely thermophilic archaebacteria; Fischer F et al.; Several types of extremely thermophilic archaebacteria have recently been isolated from solfataric water holes, hot springs and hot sea floors . It has been shown that some of them can live using sulphur respiration of reduced carbon substrates as a source of energy, a type of metabolism previously described for the eubacterium Desulfuromonas . We report here that several extremely thermophilic archaebacteria can live with carbon dioxide as their sole carbon source, obtaining energy from the oxidation of hydrogen by sulphur, producing hydrogen sulphide . They are thus capable of a new type of anaerobic, purely chemolithoautotrophic metabolism, a possible primaeval mode of life.

J Appl Bacteriol, 1983 Feb, 54(1), 115 - 25
Development of a blood-free Campylobacter medium: screening tests on basal media and supplements, and the ability of selected supplements to facilitate aerotolerance; Bolton FJ et al.; The capacity of six basal media to support the growth of thermophilic campylobacters was tested . The most successful was Nutrient Broth No . 2 (Oxoid) solidified with New Zealand agar but it gave at best only a 9% recovery rate . Various blood products, iron compounds, detoxifying agents, reducing agents, growth stimulants and an antimetabolite were added to the selected basal medium and counts of inoculated organisms were compared with counts on basal medium containing 5% lysed horse blood . Of 22 supplements tried only blood, Fildes' peptic digest of blood, heamatin, iron salts, charcoal, sodium metabisulphite and sodium pyruvate greatly improved the basal medium . The ability of these supplements used singly and in combinations to facilitate aerotolerance of campylobacters was investigated . Two aspects of aerotolerance were tested; (a) the ability of the supplements to sustain the viability of campylobacters seeded onto culture plates left on the bench for up to 6 h before microaerobic incubation; and (b) the ability of the supplements to facilitate the growth of campylobacters at increasing oxygen tension (6, 10 and 17% oxygen) . A combination of 0.4% charcoal, 0.025% ferrous sulphate and 0.025% sodium pyruvate was found to be as effective as blood in both tests.

J Biochem (Tokyo), 1983 Feb, 93(2), 503 - 11
Purification and properties of adenosine 5'-triphosphate-dependent deoxyribonuclease from Thermus thermophilus HB8; Watanabe N et al.; An ATP-dependent DNase has been purified from Thermus thermophilus HB8 by a procedure involving streptomycin precipitation, DEAE-cellulose chromatography, Sephadex G-200 gel filtration and heparin-agarose affinity chromatography . ATP-dependent DNase activity was separated into two distinct peaks, Peak A and Peak B, by heparin-agarose affinity chromatography . Each peak fraction was further purified by ATP-agarose affinity chromatography . Peak A and Peak B were eluted from an ATP-agarose column at 0.14 M and 0.28 M KCl, respectively, each as a single peak . Both enzyme activities require ATP and Mg2+ for the degradation of double- and single-stranded DNAs, and degrade denatured DNA about 1.5 times faster than native DNA . The two peaks are optimally active at 69 degrees C and have similar optimal pH ranges from 8.2 to 9.2 . The two purified peaks were unstable on storage at -20 degrees C, but were remarkably stabilized by addition of 0.4 mg/ml bovine serum albumin . Ammonium sulfate strongly inhibits the activities of both peaks . The molecular weights of Peak A and Peak B are about 170,000 as estimated by glycerol gradient sedimentation . The average chain lengths of denatured DNA produced by Peak A and Peak B were 4.2 and 3.6, respectively, and the products were terminated by 5'-phosphoryl and 3'-hydroxyl groups . The limit-digested products of denatured DNA produced by Peak B consist of mono-, di-, tri-, tetra-, and pentanucleotides along with some larger fragments . The mode of action of both activities is processive and Peak A does not attack double-stranded circular DNA.

J Biochem (Tokyo), 1983 Feb, 93(2), 461 - 8
Investigation of actin in Tetrahymena cells . A comparison with skeletal muscle actin by a devised two-dimensional gel electrophoresis method; Hirabayashi T et al.; Total protein constituents of Tetrahymena thermophila strain B1868 III were studied by two-dimensional agarose-polyacrylamide gel electrophoresis to detect actin among the constituents . In the attempts to prepare a whole-cell extract of Tetrahymena, it was found that protease activity in the extract was so high that high molecular components were quickly digested with the endogenous protease into small peptides unless the homogenization and heat-treatment in a sodium dodecylsulfate solution were performed within 5 s . It was eventually found that employment of 8 M guanidine hydrochloride (HCl) in the homogenization of cells perfectly prevented the degradation of protein components, even through a long preparation procedure . A devised two-dimensional agarose-polyacrylamide gel electrophoresis of the guanidine HCl extract gave a 'protein map' on which most proteins were located in their respective positions, including proteins with more than 200,000 mol . wt . Addition of rabbit skeletal muscle actin on the protein map revealed that no protein with isoelectric point and molecular weight identical with those of the actin was contained in the whole Tetrahymena extract, suggesting that Tetrahymena actin may have characteristics far different from those of skeletal muscle actin.

Lab Anim Sci, 1983 Feb, 33(1), 56 - 9
Preliminary findings on the use of protozoa (Tetrahymena thermophila) as models for ocular irritation testing in rabbits; Silverman J; The ciliated protozoan Tetrahymena thermophila (ATCC 30008) was used as an indicator organism in the development of an in vitro test to replace in vivo testing of chemicals in the eyes of rabbits . Fifty microliters of Tetrahymena suspension were mixed with fifty microliters of varying dilutions of test chemicals . After 2 minutes, a bacteriological loopful of the mixture was examined microscopically to evaluate the motility of the organism . The minimum dilution allowing nearly 100% typical cell motility was recorded as was the maximum dilution causing nearly 100% cell death . The reciprocals of these two dilutions were added together, and the higher this number, the more toxic the compound . The results were compared to published reports of rabbit eye irritancy studies . The preliminary in vitro testing indicated a close correlation with in vivo testing with many, but not all chemicals tested . The incidence of false negatives was minimal.

Appl Environ Microbiol, 1983 Feb, 45(2), 381 - 3
House flies (Musca domestica) as possible vectors of Campylobacter fetus subsp . jejuni; Rosef O et al.; A total of 161 strains of Campylobacter fetus subsp . jejuni were isolated from house flies (Musca domestica) . The carrier rates detected were 50.7% in flies captured on a chicken farm and 43.2% in flies from a piggery . The relative prevalences of Campylobacter coli, C . jejuni, and nalidixic acid-resistant thermophilic campylobacters were 90.1, 6.2, and 3.7%, respectively . The results indicate that flies may play a linking role in the epidemiology of Campylobacter infection in humans by transmitting campylobacters from animals to human food.

Biochimie, 1983 Feb, 65(2), 149 - 56
Purification and characterization of an endoglucanase from a newly isolated thermophilic anaerobic bacterium; Creuzet N et al.; An endoglucanase (1,4-beta-D-glucan glucanohydrolase, EC 3.2.1.4) from a new cellulolytic thermophilic bacterium was purified to apparent homogeneity after being separated from a xylanase . Little carbohydrate was associated with the endoglucanase . A molecular weight of 91,000 and 99,000 was determined by SDS-polyacrylamide gel electrophoresis and by gel filtration of the native enzyme on Ultrogel ACA 34 . The optimal pH was approximately 6.4 and the enzyme was isoelectric at pH 3.85 . The enzyme was found highly thermostable: it retained 50 per cent of its activity after 1 hour at 85 degrees C . Hydrolysis of CMC took place with a rapid decrease in viscosity but a slow liberation of reducing sugars, indicating endo-enzyme activity . It showed little capacity to hydrolyze highly ordered cellulose . Cellobiose inhibited the activity of the endoglucanase . None of the metal ions tested stimulated the activity . The enzyme was completely inactivated by 1 mM Hg2+ and was inhibited by thiol reagents.

Exp Cell Res, 1983 Feb, 143(2), 461 - 7
An amicronucleate mutant of Tetrahymena thermophila; Kaney AR et al.; A stable amicronucleate strain of Tetrahymena thermophila was isolated following nitrosoguanidine mutagenesis . The mutant has the same growth rate and viability as the micronucleate parent strain, and has no micronucleus detectable by chromatin-specific staining in vegetative growth or during conjugation . The mutant pairs with normal efficiency with cells of complementary mating type . Matings of the mutant with aneuploid strains which lose their micronucleus during meiosis produced cell pairs yielding one viable and one inviable cell . The mutant receives a micronucleus from a normal mating partner, but this micronucleus is lost by the mutant cells within two hundred generations.

Tsitologiia, 1983 Feb, 25(2), 210 - 3
{Comparison of the heat resistance of ATP-hydrolyzing enzymes in 2 species of frogs}; Pisareva LN et al.; A comparison was made between the heat resistance levels of two enzymatic preparations: the actomyosinic and the transport ATPases of two species of frogs (grass and lake frogs) differing in their thermophilia . The interspecies differences in the heat resistance were found to be 6 degrees C for the actomyosinic and 3 degrees C for the transport ATPase in favour of the more thermophilic lake frog . In both species the myosinic ATPase is more sensitive to heat than the transport one . The high heat resistance of the transport ATPase is due to a higher threshold of its sensitivity to temperature.

Philos Trans R Soc Lond B Biol Sci, 1983 Jan 26, 300(1100), 249 - 61
Large-scale purification of enzymes; Bruton CJ; Many standard procedures for the purification of proteins in the laboratory do not readily lend themselves to scaling up, whereas, on the other hand, some techniques relatively unsatisfactory in the laboratory are much more effective on a large scale . When producing gram or kilogram quantities of enzymes for use over an extended period, the storage properties and general tractability of the purified products become increasingly important . Hence enzymes from thermophilic sources frequently have advantages over those from mesophiles . The possible economic advantages of simultaneous large-scale multi-enzyme isolation over separate individual enzyme purifications are evaluated . Batchwise adsorption and elution from ion-exchange celluloses frequently replace traditional precipitation techniques in the early stages of a large-scale purification . Dialysis is replaced by concentration, dilution and reconcentration with the use of hollow-fibre ultrafiltration equipment . Antiphonally direct scaling-up of column chromatographic procedures is usually possible . Modifications to column geometry to maximize flow rates are often desirable but purification factors and recoveries comparable with those obtained on the laboratory scale can be achieved relatively easily . Classical affinity chromatographic techniques have not proved so amenable to large-scale work, mainly because of the enormous expense and rather short life of the matrices . However, the quasi-affinity chromatography afforded by the triazine dye conjugates has proved of great benefit . The materials are cheap to prepare . The coupling procedures are both simple and rapid and do not involve the use of noxious chemicals such as cyanogen bromide . Moreover the triazine linkage is more stable under a variety of conditions than the isourea formed in cyanogen bromide coupling . Considerable further exploitation of these versatile matrices is expected.

Biochemistry, 1983 Jan 18, 22(2), 484 - 9
Glycosylation, ADP-ribosylation, and methylation of Tetrahymena histones; Levy-Wilson B; We have examined some of the postsynthetic modifications that occur in macronuclear histones from Tetrahymena thermophila . When purified macronuclei are incubated with {32P}NAD+, histones H1, H2A, H2B, and H3 are ADP-ribosylated . Furthermore, histones H1, H2A, H2B, and H3 contain fucose and mannose residues as evidenced by the incorporation of {3H}fucose and by the specific binding to these proteins of gorse seed lectin and concanavalin A . Finally, our studies on incorporation of methyl groups into histones show that histone H2A, together with the related nonhistone protein A24, is methylated in Tetrahymena.

Biochim Biophys Acta, 1983 Jan 12, 742(1), 197 - 205
Ornithine decarboxylase activity from an extremely thermophilic bacterium, Clostridium thermohydrosulfuricum . Effect of GTP analogues on enzyme activity; Paulin L et al.; The activity of ornithine decarboxylase has been detected for the first time in extracts of a thermophilic bacterium, Clostridium thermohydrosulfuricum . The temperature optimum of the thermoresistant ornithine decarboxylase was 55 degrees C and the pH optimum was 7.5 . It required pyridoxal phosphate and a thiol (dithiothreitol) for activity . The activity of the enzyme was closely connected to the growth of the thermophilic bacteria, since the activity was highest during the logarithmic growth . The enzyme was not inhibited (in contrast to the enzyme from Escherichia coli) by putrescine, spermidine or other naturally occurring polyamines . When the effect of GTP and a number of GTP analogues was tested on the activity of the enzyme, it was observed that GTP or dGTP was necessary for the full activity . The modification of either the purine base or 5'-phosphate chain of GTP leads to a stimulation smaller than that caused by GTP . Modification of the 3'-carbon of the ribose part of GTP (magic spot I and II of Cashel and Gallant, Nature 221 (1969) 838-841) caused a distinct inhibition of the enzyme activity, indicating that ornithine decarboxylase contains at least two domains for binding of GTP . The enzyme was inhibited irreversibly by high concentrations (50 mM) of difluoromethylornithine . Extracts of the bacterium contained also arginine decarboxylase, but its activity was always very much lower than that of ornithine decarboxylase . The activity of arginine decarboxylase was inhibited irreversibly by difluoromethylarginine, which is an irreversible suicide inhibitor of bacterial arginine decarboxylase (Kallio, A., McCann, P.P . and Bey, P . (1981) Biochemistry 20, 3163-3166).

Biochemistry, 1983 Jan 4, 22(1), 98 - 102
Separation and comparison of 2-thioribothymidine-containing transfer ribonucleic acid and the ribothymidine-containing counterpart from cells of Thermus thermophilus HB 8; Watanabe K et al.; For the extreme thermophile Thermus thermophilus HB 8, a positive correlation was observed among the growth temperatures of the cells, the melting temperature, and the 2-thioribothymidine (s2T) content of tRNA extracted from cells grown at various temperatures {Watanabe, K., Shinma, M., Oshima, T., & Nishimura, S . (1976) Biochem . Biophys . Res . Commun . 72, 1137-1144} . On the basis of these observations, studies were carried out from which the following results were obtained . (1) Both RNase T1 and U2 digestions of tRNA gave only four fragments containing s2T or T: s2T psi CGp, s2T psi CAp, T psi CGp, and T psi CAp . For the different growth temperatures, the ratio of the content of s2T psi CGp plus s2T psi CAp to that of T psi CGp plus T psi CAp was almost the same as that of the s2Tp to Tp content reported previously . (2) The midpoint of the s2T-specific circular dichroism spectral change induced by heat was constant for all tRNAs extracted from cells grown at various temperatures, suggesting that the s2T-containing tRNAs melt at about the same temperature, which is independent of the growth temperature of cells . (3) s2T-containing tRNA was separated from the T-containing counterpart quantitatively by a specific modification of s2T with bromoaceto-2,4-dinitroanilide followed by BD-cellulose column chromatography . The molar ratio of the s2T- and T-containing tRNAs was also similar to that of s2Tp to Tp as mentioned above . These results demonstrate that T . thermophilus cells have both s2T- and T-containing tRNAs, whose relative content is controlled by the growth temperature . This phenomenon may be necessary to enable the thermophile to adapt to higher temperatures.

Biochemistry, 1983 Jan 4, 22(1), 85 - 93
Localization of the elongation factor Tu binding site on Escherichia coli ribosomes; Rychlik W et al.; Fluorescent techniques were used to study binding of peptide elongation factor Tu (EF-Tu) to Escherichia coli ribosomes and to determine the distances of the bound factor to points on the ribosome . Thermus thermophilus EF-Tu was labeled with 3-(4-maleimidylphenyl)-4-methyl-7-(diethyl-amino)coumarin (CPM) without loss of activity . In the presence of Phe-tRNA and a nonhydrolyzable analogue of GTP, 70S ribosomes bind the CPM-EF-Tu {Kb = (3 +/- 1.2) X 10(6) M-1} causing a decrease of CPM fluorescence . Binding of CPM-EF-Tu to 50S subunits was at least 1 order of magnitude lower than with 70S ribosomes, and binding to 30S subunits could not be detected . Reconstituted 70S ribosomes containing either S1 labeled with fluoresceinmaleimide or ribosomal RNAs labeled at their 3' ends with fluorescein thiosemicarbazide were used for energy transfer from CPM-EF-Tu . The distances between CPM-EF-Tu bound to the ribosomes and the 3' ends of 16S RNA, 5S RNA, 23S RNA, and the closest sulfhydryl group of S1 were calculated to be 82, 70, 73, and 62-68 A, respectively.

Nutr Cancer, 1983, 5(3-4), 159 - 64
Effect of feeding fermented milk on the incidence of chemically induced colon tumors in rats; Shackelford LA et al.; The effect of feeding skim milk fermented by Streptococcus thermophilus or Lactobacillus bulgaricus on the incidence of chemically induced colon tumors was studied in rats . Weanling Fisher-344 rats were fed chow plus skim milk (SM), chow plus SM fermented by S . thermophilus, chow plus SM fermented by L . bulgaricus, or chow plus water until sacrifice at 36 weeks, or before if moribund . Colon tumors were induced by s.c . injections of 1,2-dimethylhydrazine hydrochloride during weeks 3 through 22 . The control (chow + water) group received saline injections . The survival rate of the rats fed fermented milks was significantly higher than that of the rats fed nonfermented milk . The latter had a significantly higher incidence of ear-duct tumors than the rats receiving fermented milk . The percentage of rats showing colon tumors was similar among all three experimental groups . The control group did not have any tumors . The rats receiving fermented milk had a significantly higher incidence of small-intestine tumors than those receiving nonfermented milk . The rats on S . thermophilus milk had the lowest percentage of malignant colon tumors of the three experimental groups . Results indicated that the feeding of fermented milks altered the metabolism of 1,2-dimethylhydrazine and shifted the target organ from the ear duct to the small intestine . In addition, the colon tumor distribution for the fermented-milk groups appeared to shift toward the anus.

Nucleic Acids Symp Ser, 1983, (12), 217 - 20
An algorithm for the bonding-probability map of nucleic acid secondary structure; Suyama A et al.; We report a more efficient and well-defined algorithm for predicting a secondary structure of single-stranded nucleic acid from a primary nucleotide sequence . Using this algorithm, one- and two-dimensional bonding-probability maps of 5S rRNA of thermus thermophilus HB8 were calculated . These maps well express the stability of the secondary structure.

Nucleic Acids Symp Ser, 1983, (12), 173 - 6
Synthesis of model compounds for the interaction between modified nucleic acid bases; Higashii T et al.; In order to study the stacking interaction of modified nucleic acid bases, which is assumed to be responsible for the anomalous thermostability of thermophile tRNA, several model compounds 1-6 are synthesized . On the basis of their hypochromicities, the interaction between bases are discussed.

Nucleic Acids Symp Ser, 1983, (12), 153 - 4
Aminoacyl-tRNA synthetases from an extreme thermophile, Thermus thermophilus HB8; Kohda D et al.; Thermostable aminoacyl-tRNA synthetases specific to Val, Ile, Met and Glu were purified from an extreme thermophile, Thermus thermophilus HB8 . As for the subunit compositions and molecular weights, these four aminoacyl-tRNA synthetases are similar to the corresponding enzymes from E . coli and B . stearothermophilus . Val-tRNA, Ile-tRNA and Met-tRNA synthetases from T . thermophilus have two tightly bound zinc ions, whereas Glu-tRNA synthetase does not . The amino acid compositions and secondary structures of Val-tRNA, Ile-tRNA and Met-tRNA synthetases are quite similar to one another . The conformational transition involving the anticodon of E . coli tRNAGlu as complexed with Glu-tRNA synthetase from T . thermophilus is necessary for the aminoacylation activity.

Int J Vitam Nutr Res, 1983, 53(4), 394 - 7
{Microbiological assay of pantothenic acid in rat liver using Tetrahymena}; Lhuissier M; Microbiological Assay of Pantothenic Acid in Rat Liver using Tetrahymena . The quantitative determination of total pantothenic acid in rat liver using Tetrahymena was carried out in the same way as a microbiological assay . The strain selected was Tetrahymena thermophila grown on rat liver . Inoculum was obtained from a commercial medium . The assay medium consisted of a dry powder to be rehydrated and heated to boiling, and then adjusted to pH 7 . Liver pantothenic acid was extracted by autolysis . Tetrahymena use made one accustomed to a biological reagent easy to manipulate but with complex nutritional requirements . Consequently the organism should be more extensively used for nutritional or toxicological studies.

Biochem Soc Symp, 1983, 48, 133 - 46
Industrial prospects for thermophiles and thermophilic enzymes; Hartley BS et al.; Reasons for enzyme instability are discussed . Thermophiles are a promising source of more stable intracellular enzymes . This aids their purification as well as providing desirable industrial properties . The organisms themselves have advantages for high temperature fermentations, e.g . for ethanol production . Systems for cloning genes into them are under development . An example is given of genetic and physiological manipulation of a fast-fermenting Bacillus stearothermophilus to increase ethanol yields at 70 degrees C similar to those obtained with yeasts.

J Biochem (Tokyo), 1983 Jan, 93(1), 7 - 13
Heat-stable extracellular proteolytic enzyme produced by Thermus caldophilus strain GK24, an extremely thermophilic bacterium; Taguchi H et al.; Proteolytic activity was detected in the culture supernatant of a newly isolated, extremely thermophilic bacterium belonging to the genus Thermus, and tentatively named T . caldophilus sp . n . strain GK24 . The enzyme activity continued to increase for at least three days after cells reached the stationary phase of growth . Purification of the proteolytic enzyme was tried with ammonium sulfate fractionation, gel filtration, and ion exchange chromatography . The most purified enzyme fraction thus obtained appeared to be homogeneous in a chromatographic analysis, but still had seven bands of proteins on sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis . Treatment of the protease with denaturing reagents or organic solvents did not alter the chromatographic profile and the purified enzyme sample showed a large sedimentation coefficient of about 11S . The optimal pH of the hydrolytic activity of the enzyme was observed at around 7.8 for casein and 7.2 for N-carbobenzoxy-L-leucyl-L-tyrosinamide (Z-Leu-Tyr-NH2) . The enzyme was stable in the pH range of 5 to 11 for 1 day at 4 degrees C or for 1 h at 70 degrees C . The enzyme sample showed a maximal activity at 90 degrees C and had an extreme stability toward treatment by heat and denaturing reagents . The enzyme sample was inactivated almost completely by diisopropyl fluorophosphate (DFP), but not by ethylenediaminetetraacetic acid (EDTA) or ethylene glycol-bis(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA) . From these results, the enzyme seems to be a serine protease, and not to be a metallo-enzyme such as thermolysin . The enzyme also was hydrolytic active toward an ester compound, N-benzoyl-L-tyrosine ethyl ester (BTEE), but not toward N-benzoyl-L-arginine ethyl ester (BAEE).

J Membr Biol, 1983, 75(1), 45 - 56
Monolayer black membranes from bipolar lipids of archaebacteria and their temperature-induced structural changes; Gliozzi A et al.; The membrane of Caldariella acidophila, an extreme thermophilic archaebacterium, is characterized by unusual bipolar complex lipids . They consist of two nonequivalent polar heads, linked by a C40 alkylic component . The molecular organization of these lipids in the plasma membrane is still a matter of study . In this paper, we present current-voltage measurements on artificial bipolar lipid membranes, indicating that molecules are indeed organized as a covalently bound bilayer, in which each molecule is completely stretched and spans its entire thickness . Furthermore, conformational transitions of these artificial membranes (which could be formed only above 70 degrees C from a lipid/squalene dispersion) are analyzed in the 80 to 15 degrees C temperature range . Abrupt variations in capacitance and valinomycin-induced conductance seem to indicate the occurrence of at least two structural changes . Measurements are also extended to different solvent systems . Results are consistent with the picture of a monolayer bipolar lipid membrane in which few solvent molecules align themselves parallel to the lipophilic chains . The amount of solvent as well as the temperature at which conformational transitions occur, depend on the solvent system in which the lipid is dispersed.

J Biochem (Tokyo), 1983 Jan, 93(1), 225 - 34
Outer and inner membrane preparations of extreme thermophile and their physico-chemical properties; Oshima M et al.; Outer and inner (cytoplasmic) membranes were partially purified from the gram negative extremely thermophilic bacteria, Thermus thermophilus HB-8 by sucrose density gradient centrifugation . In spite of our efforts to separate them, the inner membrane fraction contained some outer membrane components as determined by enzyme assay and electrophoresis . When studied by 5DS spin labeling, the outer membranes showed a larger 2T11 value (lower fluidity) than the inner membranes, although the fatty acid compositions were similar . The inner membranes of the cells cultured at higher temperature showed a larger 2T11 value than the cells cultured at lower temperature . A similar phenomenon was observed with the TEMPO parameter of liposomal membranes . The upper break point (Th) of the inner membranes observed by spin labeling was slightly lower than the culture temperature of the cells, and the lower break point (T1) corresponded well to the lowest temperature limit of growth . The calorimetric heating curve of the inner membranes had a broader temperature range of transition than that of the liposomal membranes . The transition temperature observed by calorimetry seems to reflect the melting properties of the membrane lipids, while fatty acid spin probe probably reports the local environment of the membrane, which is more directly related to its biological function.

Eur J Biochem, 1982 Dec 15, 129(2), 429 - 36
Purification and characterization of tRNA (adenine-1-)-methyltransferase from Thermus flavus strain 71; Morozov IA et al.; tRNA (adenine-1-)-methyltransferase was isolated from the extreme thermophile Thermus flavus, strain 71 . It was purified about 2000-fold by ammonium sulfate fractionation and affinity chromatography on tRNA bound to aminohydroxybutylcellulose via its oxidized 3' end . The purified protein preparation is free of nuclease and aminoacyl-tRNA synthetase activity and contains no more than 4% of tRNA (guanine-7-)methyltransferase activity . The only activity of the enzyme is to methylate A58 in the T psi loop of tRNA . Out of the eight purified tRNAs examined, only yeast tRNATrp was not utilized as a substrate . The enzyme is highly thermostable . It is most active at 75 degrees C . tRNA (adenine-1-)-methyltransferase has a Km of 0.4-0.5 microM for tRNA2Gln from Escherichia coli and a Km of 6 microM for S-adenosyl-L-methionine.

J Anim Sci, 1982 Dec, 55(6), 1293 - 302
Nutritive value of methane fermentation residue in diets fed to feedlot steers; Harris JM et al.; Nutritive value of the methane fermentation residue (MFR) from the effluent of a large scale thermophilic methane generator was determined in diets fed to feedlot steers . The MFR contained 22.2% dry matter and 21.9% crude protein (dry basis) . Two diets containing 10.6% (dry basis) MFR were formulated using the Urea Fermentation Potential (UFP) system such that in one diet N was in excess (-1.6 UFP) while in the other diet energy was in excess (+2.6 UFP) . These two diets were compared in a California Net Energy trial with a feedlot diet (-.3 UFP) containing the same ingredients except the MFR . Six steers were fed in a replicated 3(2) Latin square metabolism trial and 70 steers were fed in a 118-d comparative-slaughter, feedlot trial . Digestibilities of dry matter, organic matter, crude protein, acid detergent fiber, ash, total digestible nutrients (TDN) and metabolizable energy were depressed (all P less than .05) in the MFR-containing diets . Steers fed the MFR-containing diets had lower (P less than .05) rates of gain and increased (P less than .05) feed requirements per unit gain . Net energies for maintenance and gain were slightly lower for the MFR-containing diets than the control diet . Crude protein digestibility for the MFR calculated by difference, for the -UFP and the +UFP diets were 37.8 and 50.7%, while corresponding values for TDN were 28.8 and 12.8%, respectively . Concentrations of potentially toxic elements in kidney, liver and muscle as well as flavor and tenderness of steaks were not affected by feeding MFR.

J Lipid Res, 1982 Dec, 23(9), 1301 - 7
The effect of temperature on glyceryl ethers in Tetrahymena pyriformis W; Lund-Katz S et al.; The effect of temperature on the ether content of the glycerophospholipids of Tetrahymena pyriformis W was examined . The only ether detected was 1-O-hexadecyl glycerol (alpha-chimyl alcohol) . The data provide evidence that the class 1-O-alkyl-2-acyl-sn-glycero-3-(2-aminoethyl)-phosphate (1-alkyl PsE), in addition to the previously reported 1-O-alkyl-2-acyl-sn-glycero-3-(2-aminoethyl)-phosphonate (1-alkyl PnE) and 1-O-alkyl-2-acyl-sn-glycero-3-phosphorylcholine (1-alkyl PC), exists in this ciliate species . A comparison was made of the ether content of the glycerophospholipids from cells grown at 15 degrees and 28.5 degrees C . An elevation in the amount of ether was noted in all glycerophospholipids at the lower temperature with the largest proportional change in 1-alkyl PsE . Tetrahymena species have a high gamma-linolenic acid content in the sn-1 position of the glycerophospholipids in addition to the usual saturated acids and ether . The replacement at low temperature of gamma-linolenic acid by a saturated hydrocarbon at the sn-1 position of the glycerophospholipids of Tetrahymena pyriformis W should increase the microviscosity of the membranes; thus, it is difficult to envision this alteration in the glycerophospholipids as an adaptive change beneficial for growth . These findings are in direct contrast to the situation in Tetrahymena thermophila where the percentage of ether glycerophospholipids increases at the expense of gamma-linolenate as the temperature rises.

J Clin Microbiol, 1982 Dec, 16(6), 995 - 9
Comparison of methods for isolation and enumeration of thermophilic actinomycetes from dust; Treuhaft MW et al.; Thermophilic actinomycetes are the primary sensitizing agents in farmer's lung disease . We compared dilution pour-plate and spread-plate methods for their usefulness in enumerating thermophilic actinomycetes in moldy silage dust and evaluated the ability of a nonquantitative gravity settling technique to recover thermophilic actinomycetes from moldy silage . Spread plates and pour plates yielded similar estimates of total thermophiles . Higher counts were observed on spread plates (P less than 0.05) for Thermoactinomyces candidus, Micropolyspora faeni, and Saccharomonospora viridis . M . faeni and S . viridis were less efficient than T . candidus in breaking through the agar of pour plates to form colonies which could be identified . Coefficients of variability were less than 10% for the two methods . The relative proportion of organisms recovered by the settling method correlated well with that recovered on spread plates for M . faeni (r = 0.79), S . viridis (r = 0.88), and Thermomonospora spp . (r = 0.79), but not well for T . candidus (r = 0.28) . When sophisticated air-sampling equipment is not available, dilution spread plates of dust washings provide a reproducible method for enumerating a broad range of thermophilic actinomycetes of interest . The gravity settling method is a simple, rapid alternative when isolation is all that is required.

J Biochem (Tokyo), 1982 Dec, 92(6), 1823 - 32
A new aspect of a restriction endonuclease Tth111 I . It has a degenerated specificity (Tth111 I); Shinomiya T et al.; We previously reported that Thermus thermophilus 111 contained two restriction enzymes, Tth111 I and Tth111 II . The former does not cleave phi X174RFDNA and the latter does . We have now found another endonuclease activity able to cleave phi X174RFDNA in the cell extract of T . thermophilus 111 . The protein with this activity was purified in a homogeneous state by chromatography on cellulose phosphate, heparin-Sepharose 4B and hydroxylapatite, successively . However, this endonuclease activity was always accompanied with Tth111 I activity during the purification procedure and the purified protein also showed a strong Tth111 I activity, suggesting that the Tth111 I activity and the phi X174RFDNA-cleaving activity reside in a single molecule . The phi X174RFDNA-cleaving activity was enhanced more strongly with Mn2+ than with Mg2+ and seemed to be attributable to a relaxed specificity of Tth111 I activity as seen in the cases of EcoRI* and BamHI* Thus we designated the phi X174RFDNA-cleaving activity Tth111 I* . The molecular weight of the protein with both Tth111 I and Tth111 I* activities was determined to be about 76,000 by gel filtration on a Sephadex G-100 column and 39,000 by SDS-polyacrylamide gel electrophoresis, suggesting the enzyme to be a dimer consisting of identical polypeptide chains . The phi X174RFDNA sequences surrounding Tth111 I* cuts were determined by the chain terminator method of Sanger et al . The results confirmed that Tth111 I* recognized a degenerated form of the Tth111 I recognition sequence, i.e., a sequence such that one of the specified nucleotides in the Tth111 I recognition sequence, 5'GACNNNGTC3', was substituted with N (N stands for any of A, G, C, and T), such as 5'NACNNNGTC3', 5'GACNNNNTC3', 5'GACNNNGNC, and so on (arrows indicate cleavage sites).

J Biochem (Tokyo), 1982 Dec, 92(6), 2043 - 6
Nanosecond fluorometric investigation of hydrodynamic properties of adenosine triphosphatase from thermophilic bacterium PS3; Kinosita K Jr et al.; The soluble portion (TF1) of proton-translocating ATPase from thermophilic bacterium PS3 was labeled with a fluorescent dye N-(1-pyrene)maleimide . The decay of fluorescence anisotropy of the adduct showed that TF1 in aqueous solution was characterized by a volume of equivalent sphere of 1,120 nm3 . This value is 2.4 times the volume calculated from the molecular weight and partial specific volume, indicating a non-spherical shape and/or extensive hydration . A prolate ellipsoid with an axial ratio of 2 to 3 is suggested as a first approximation of the shape of hydrated TF1 . The presence or absence of ATP, ADP, or Mg2+ did not alter the volume of the equivalent sphere appreciably; the probable conformational change of TF1 induced by these ligands does not lead to a gross alteration of its hydrodynamic properties.

J Biol Chem, 1982 Nov 10, 257(21), 12600 - 4
Proton transport by cytochrome c oxidase from the thermophilic bacterium PS3 reconstituted in liposomes; Sone N et al.; Cytochrome oxidase from the thermophilic bacterium PS3 which contains three types of polypeptide subunits are reconstituted into liposomes by a freeze-thaw technique . The reconstituted enzyme caused acidification of the medium during cytochrome c oxidation with a stoichiometry of up to 0.8 H+/e . Uptake of K+ ions in the presence of valinomycin occurred with a stoichiometry between 1.5 and 2 K+/e . Dicyclohexylcarbodiimide inhibited the acidification and decreased the stoichiometry of K+ ion uptake to about 1 K+/e . This bacterial oxidase thus appears to be a proton pump with properties similar to the mitochondrial enzyme.

J Biol Chem, 1982 Nov 10, 257(21), 12489 - 92
Mössbauer study of a bacterial cytochrome oxidase: cytochrome c1aa3 from Thermus thermophilus; Kent TA et al.; Cytochrome c1aa3 from Thermus thermophilus has optical and EPR properties similar to bovine cytochrome c oxidase . We have studied 87Fe-enriched samples with Mossbauer spectroscopy in the fully oxidized and fully reduced states and in the oxidized state complexed with cyanide . The cytochromes a and c1 yielded spectra quite similar to those reported for the cytochromes c and b5; in the oxidized state the spectra reflect noninteracting, low spin ferric hemes, whereas the a- and c1-sites of the reduced enzyme are typical of low spin ferrous hemochromes . The spectra of the reduced enzyme show that reduced cytochrome a3 is high spin ferrous, with Mossbauer parameters quite similar to those of deoxymyoglobin . Upon addition of cyanide to the oxidized enzyme, the a3-site exhibits in the absence of an applied magnetic field and at temperatures down to 1.3 K a quadrupole doublet with parameters typical of low spin ferric heme-CN complexes . The low temperature spectra taken in applied magnetic fields show that the electronic ground state of the a3-CN complex has integer electronic spin, suggesting ferromagnetic coupling of the low spin ferric heme (S = 1/2) to Cu2+ (S = 1/2) to yield as S = 1 ground state . We have examined the oxidized enzyme from two different preparations . Both had good activity and identical optical and EPR spectra . The Mossbauer spectra, however, revealed that the a3-site had a substantially different electronic structure in the two preparations . Neither configuration had properties in accord with the widely accepted spin-coupling model proposed for the bovine enzyme.

J Gen Microbiol, 1982 Nov, 128 (Pt 11), 2515 - 22
Deoxyribonucleic acid sequence relatedness between thermophilic members of the genus Campylobacter; Belland RJ et al.; The genomic relatedness among 28 catalase-positive Campylobacter strains was assessed by determination of DNA base composition, by DNA:DNA hybridization followed by S1 endonuclease digestion of single-stranded DNA, and by determining the thermal stability of homologous and heterologous double-stranded DNA . The catalase-positive Campylobacter strains were shown to comprise 3 species, C . coli, C fetus and C . jejuni; each included the type strain (respectively, CIP 7080, CIP 5396 and CIP 702).

Biokhimiia, 1982 Nov, 47(11), 1785 - 91
{Isolation and properties of DNA polymerase from the extreme thermophilic bacterium Thermus ruber}; Kaledin AS et al.; A DNA-polymerase from the external thermophylic bacteria Thermus ruber has been isolated . A six-step purification procedure resulted in an electrophoretically homogeneous enzyme preparation with m . w . of 70 000 . The isolated enzyme is thermostable and has a temperature optimum on DNA templates at 70 degrees and that on RNA templates at 50 degrees . The enzyme does not contain contaminant endo- and exonuclease activities . The maximal activity of the enzyme requires the presence of template, four deoxyribonucleoside triphosphates, monovalent and divalent cations in the incubation mixture . The catalytic activity of the enzyme on various templates of DNA- and RNA-types has been investigated . A comparative physico-chemical study of this DNA-polymerase and an analogous enzyme isolated earlier from the external thermophylic bacteria Thermus aquaticus YT-1 has been carried out.

Cell, 1982 Nov, 31(1), 147 - 57
Self-splicing RNA: autoexcision and autocyclization of the ribosomal RNA intervening sequence of Tetrahymena; Kruger K et al.; In the macronuclear rRNA genes of Tetrahymena thermophila, a 413 bp intervening sequence (IVS) interrupts the 26S rRNA-coding region . A restriction fragment of the rDNA containing the IVS and portions of the adjacent rRNA sequences (exons) was inserted downstream from the lac UV5 promoter in a recombinant plasmid . Transcription of this template by purified Escherichia coli RNA polymerase in vitro produced a shortened version of the pre-rRNA, which was then deproteinized . When incubated with monovalent and divalent cations and a guanosine factor, this RNA underwent splicing . The reactions that were characterized included the precise excision of the IVS, attachment of guanosine to the 5' end of the IVS, covalent cyclization of the IVS and ligation of the exons . We conclude that splicing activity is intrinsic to the structure of the RNA, and that enzymes, small nuclear RNAs and folding of the pre-rRNA into an RNP are unnecessary for these reactions . We propose that the IVS portion of the RNA has several enzyme-like properties that enable it to break and reform phosphodiester bonds . The finding of autocatalytic rearrangements of RNA molecules has implications for the mechanism and the evolution of other reactions that involve RNA.

J Biol Chem, 1982 Oct 10, 257(19), 11395 - 404
Structural studies of 5 S ribosomal RNAs from a thermophilic fungus, Thermomyces lanuginosus . A comparison of generalized models for eukaryotic 5 S RNAs; Wildeman AG et al.; The cytoplasmic ribosomes of the thermophilic fungus Thermomyces lanuginosus contain two types of 5 S RNA . The nucleotide sequence for approximately 80% of the molecules is (pp)pA-C-A-U-G-C-G-A-C-C-A-U-A-G-G-G-U-G-U-G-G-A-A-A-A-C-A-G-G-G-C-U-U-C-C-C-G-U-C-C-G-C-U-C-A-G-C-C-G-U-A-C-U-U-A-A-G-C-C-A-C-A-C-G-C-C-G-G-C-U-G-G-U-U-A-G-U-A-G-U-U-G-G-G-U-G-G-G-U-G-A-C-C-A-C-C-A-G-C-G-A-A-U-C-C-C-A-G-C-U-G-U-U-G-C-A-U-G-UOH . The remainder contains two nucleotide substitutions, C19 and G60, which preserve base complementarity . The secondary structure was probed using partial T1, pancreatic, and S1 nuclease digestion under a variety of ionic and temperature conditions and fragments were analyzed by rapid gel sequencing techniques . The results support the Y-shaped secondary structure model originally proposed by Nishikawa, K., and Takemura, S . (1974) FEBS Lett . 40, 106-109, for eukaryotic 5 S RNAs . When the thermal denaturation profile was compared with that of the yeast 5 S RNA, the thermophilic RNA exhibited not only a higher Tm but also an unusual decline in absorbency at moderate temperatures . This suggests that a functionally important structure may be maintained only at higher temperatures.

Mol Cell Biochem, 1982 Oct 1, 48(1), 48 - 58
Glucose regulation of specific gene expression is altered in a glucokinase-deficient mutant of Tetrahymena; Lavine JE et al.; Expression of the galactokinase gene in Tetrahymena thermophila can be repressed by glucose, glucose analogs, and epinephrine, each apparently acting through increased intracellular levels of adenosine 3':5'-cyclic monophosphate (cAMP) (1) . To characterize further the initial steps in the control of galactokinase gene expression by glucose, we have analyzed mutants which are defective in the metabolism of this sugar; these mutants were selected for their resistance to the glucose analog, 2-deoxyglucose (2) . In one such mutant that is deficient in glucokinase, the synthesis of galactokinase is totally resistant to repression by glucose or its analogs, while repression by exogenous catecholamines or dibutyryl cAMP is unaffected . Radiochromatographic analyses of extracts of wild-type cells incubated with {14C}-deoxyglucose reveal intracellular conversion to several deoxyglucose metabolites, principally deoxyglucose-6-P and smaller amounts of deoxyglucose-1-P and 2-deoxygluconate; extracts of glucokinase-deficient cells prepared in a similar manner contain only trace amounts of deoxyglucose-6-P . The glucose analog 3-O-methylglucose, which is transported but not phosphorylated in wild-type cells, also cannot maintain repression of galactokinase . These results establish that the transport and subsequent phosphorylation of glucose are required for glucose-initiated repression of galactokinase gene expression, possibly acting by modulation of catecholamine or cyclic AMP levels . Additionally, we show unequivocally that: (a) cells containing derepressed levels of galactokinase are repressed upon the addition of glucose by inhibition of the synthesis of new enzyme and dilution of preformed enzyme concomitant with cell division, rather than through selective inactivation or degradation of galactokinase; and (b) glycerol kinase, glucokinase and fructokinase activities also are repressed by glucose in wild-type Tetrahymena, indicating that the glucose repression phenomenon is pleiotropic . Because the glucose repression of the synthesis of each of these enzymes is abolished in cells deficient in glucokinase, the regulatory mechanisms elucidated for repression of galactokinase synthesis are likely to be of wide significance.

J Hyg (Lond), 1982 Oct, 89(2), 191 - 4
The occurrence of Campylobacter jejuni in dog faeces from a public park; Wright EP; Dog faeces collected from a public park were cultured on selective media for Campylobacter spp . Campylobacter jejuni was isolated from 12 (4.6%) of 260 specimens . In contrast Salmonella spp . were found in only three (1.2%) . Six of the 12 isolates were nalidixic acid-resistant thermophilic campylobacters (NARTC), whereas during the same period of study none were found among human isolates . Most of the campylobacter positive faeces were found during June and July . Dog faeces deposited in public places constitute only a small potential source of infection by this organism.

Can J Microbiol, 1982 Oct, 28(10), 1181 - 8
{Peptide hydrolases of lactobacilli of the Thermobacterium group . I . Demonstration of these activities in Lactobacillus helveticus, L . acidophilus, L . lactis and L . bulgaricus}; El Soda M et al.; The intracellular peptide hydrolase activities of Lactobacillus helveticus, L . acidophilus, L . lactis, and L . bulgaricus were determined using various aminopeptidase, dipeptidase, and carboxypeptidase substrates in addition to casein and whey protein fractions . The different activities were then separated using disc gel electrophoresis . Each bacterium had aminopeptidase activity towards various amino acid beta-naphthylamides and dipeptides . The four species also showed bands of true dipeptidase activities on a large number of dipeptides . Intracellular enzymes from thermophilic lactobacilli also hydrolysed the whey proteins (alpha-lactalbumin and beta-lactoglobulin) . From the results of electrophoresis on beta-casein and alpha s1-casein it was shown that beta-casein was totally hydrolysed by L . lactis while it was only partially hydrolysed by the intracellular enzymes of L . acidophilus and L . bulgaricus . On the other hand, alpha s1-casein was only partially hydrolysed by L . helveticus, L . lactis, and L . bulgaricus.

J Biol Chem, 1982 Sep 10, 257(17), 9913 - 4
A pentaamine is present in an extreme thermophile; Oshima T; A pentaamine was found in cells of an extreme thermophile, Thermus thermophilus, grown at 80 degrees C . The chemical structure was determined to be 1,15-diamino-4,8,12-triazapentadecane, NH2(CH2)3NH(CH2)3NH(CH2)3NH(CH2)3NH2 . A trivial name "caldopentamine" is proposed for the new naturally occurring polyamine.

Med J Aust, 1982 Sep 4, 2(5), 244 - 6
Hypersensitivity pneumonitis in a mouldy house; Saltos N et al.; Two cases of hypersensitivity pneumonitis, one confirmed by histopatholgy, are described . The patients were members of different families and developed the disease as occupants, at separate times, of the same inner-city dwelling . We believe the disorder resulted from exposure to thermophilic microorganisms prevalent in their domestic environment . Both patients recovered after moving from this place of residence . The need for greater awareness of this variety of hypersensitivity pneumonitis is stressed.

Can J Biochem, 1982 Sep, 60(9), 847 - 53
Reorganization of unique and repetitive sequences during nuclear development in Tetrahymena thermophila; Brunk CF et al.; Genomic libraries of macro- and micro-nuclear DNA of the ciliate protozoan Tetrahymena thermophila were constructed in the bacteriophage vector lambda gt WES lambda B . Screening of these libraries with a probe for the repeated hexamucleotide sequence C4A2 showed many phage from the micronuclear library but few if any macronuclear sequences having homology to this probe . This is consistent with C4A2-repeating elements being present predominantly if not exclusively at or near the termini of macronuclear DNA . Sequences flanking C4A2-repeating elements were isolated from a number of purified phage and were used as hybridization probes to restriction endonuclease digested macro- and micro-nuclear DNA . These experiments revealed a repeated sequence family as well as unique sequences present only in micronuclear DNA . The repeated sequence element appears to be dispersed throughout the genome . Phage-containing individual members of this micronucleus limited sequence family were purified from the micronuclear library . Some of these phage contained sequences which hybidized to macronuclear DNA . These fragments therefore contain a "transition" region between micronucleus-limited sequences and sequences present in both nuclei.

Appl Environ Microbiol, 1982 Sep, 44(3), 757 - 60
Thermophilic biotransformations of 2,4,6-trinitrotoluene under simulated composting conditions; Kaplan DL et al.; The biotransformations of 14C-labeled 2,4,6-trinitrotoluene by thermophilic microorganisms in a compost system were determined . The reduction products identified in solvent extracts were similar to those identified in mesophilic systems . A significant percentage of the 14C-labeled products were bound to humus fractions.

Nature, 1982 Aug 26, 298(5877), 867 - 9
E . coli F1-ATPase interacts with a membrane protein component of a proton channel; Walker JE et al.; The ATP synthases of bacteria, mitochondria and chloroplasts, which use the energy of a transmembrane proton gradient to power the synthesis of ATP, consist of an integral membrane component F0--thought to contain a proton channel--and a catalytic component, F1 . To help investigate the way F0 and F1 are coupled, we have sequenced the b-subunit of the Escherichia coli F0, which seems to be the counterpart of a thermophilic bacteria F0 subunit thought to be essential for F1 binding . We report here that its sequence is remarkable, being hydrophobic around the N-terminus and highly charged in the remainder . We propose that the N-terminal segment lies in the membrane and the rest outside . The extramembranous section contains two adjacent stretches of 31 amino acids where the sequence is very similar: in the second of these stretches there is further internal homology . These duplicated stretches of the polypeptide probably fold into two alpha-helices which have many common features able to make contact with F1 subunits . Thus protein b occupies a central position in the enzyme, where it may be involved in proton translocation . It is possibly also important in biosynthetic assembly.

J Biol Chem, 1982 Aug 10, 257(15), 9114 - 8
On the phylogeny of Phycomyces blakesleeanus . Nucleotide sequence of 5 S ribosomal RNA; Andersen J et al.; The nucleotide sequence of the major 5 S ribosomal RNA from the lower fungus Phycomyces blakesleeanus has been determined . The sequence is 5' AAUCUACGGCCAUACAGAUAGUAACACACCGGAUCCCGUCUGAUCUCCGCAGUUAAGUCUCUCCUGGUAGCGUCAGUAC UAUGGUGGGGGACCACAUGGGAAUACGCUAUGUCGUAGGUU3'OH . The Phycomyces 5 S RNA sequence has invariant nucleotide positions characteristic of other eukaryotic 5 S RNAs and fits currently proposed secondary structural models . The Phycomyces of 5 S RNA shows relatively low overall sequence homology to the higher fungal (Ascomycetes) 5 S RNAs (56-60%) but shows higher sequence homology to those 5 S RNAs from Tetrahymena thermophila (68%), human KB cells (67%), and Spinacia oleracea (62%) . A comparison of individual segments of the RNA also shows that the structure of Phycomyces 5 S RNA has several major differences from structures common to the higher fungi . Positions 2-14 are homologous with those of metazoan and some protozoan 5 S RNAs . At positions 30-45, the RNA sequence is closer to metazoan 5 S RNAs than to the Neurospora of Aspergillus 5 S RNAs . The Phycomyces 5 S RNA shares similar sequences with both Aspergillus and Tetrahymena 5 S RNAs at positions 79-99 . Several other important homologies in primary and proposed secondary structures also have been observed in comparing Phycomyces 5 S RNA with animal and plant 5 S RNAs . We conclude that Phycomyces may not be as closely related phylogenetically to the Ascomycetes as previously thought.

Biochim Biophys Acta, 1982 Aug 10, 705(3), 293 - 305
Purification and some properties of an extracellular protease (caldolysin) from an extreme thermophile; Cowan DA et al.; An extracellular metal-chelator-sensitive lytic protease (assigned the trivial name caldolysin) was isolated from a Thermus-like organism, Thermus T-351 . Caldolysin was purified by affinity chromatography on Cbz-D-phenylalanine-TETA-Sepharose 4B and by gel filtration . It contained 13% carbohydrate, a single zinc atom, had a molecular weight of approx . 21,000, a pH optimum of 8 (azocasein substrate), and an isoelectric point of about 8.5 . It was capable of hydrolysing many soluble and insoluble protein substrates, including collagen and elastin . No esterase activity was detected, and small peptides (less than four amino acids) and low molecular weight chromogenic substrates were not hydrolysed . A specificity for small aliphatic amino acids on either side of the splitting point was indicated . Caldolysin lysed heat-killed Gram-negative bacterial cells, but had little effect on Gram-positive organisms . Caldolysin exhibited a very high degree of thermostability (t 1/2(80 degrees C) approximately 30 h, t 1/2(90 degrees C) = 1 h) . The stability (but not activity) was shown to be dependent on the presence of Ca2+ (t 1/2(75 degrees C, 10 mM calcium) greater than 193 h; t 1/2(75 degrees C, no calcium) = 4.8 min) . None of the other metal ions tested (Co, Zn, Sr, Mg, Ba and Cu) was as effective as calcium in conferring thermostability of EDTA-treated caldolysin . Caldolysin was stable at room temperature in moderately acid and alkaline (pH 5 to 11) buffers for periods of greater than 90 days . Little loss of enzyme activity was detected after the incubation of caldolysin at 18 degrees C in the presence of 8 M urea, 6 M guanidine hydrochloride or 1% sodium dodecyl sulphate for 24 h . At 75 degrees C, the activity half-life of caldolysin in these denaturing agents was reduced to approx . 1 h, 1 h and more than 5 h, respectively.

J Allergy Clin Immunol, 1982 Aug, 70(2), 101 - 8
Evaluation of indoor plantings as allergen exposure sources; Burge HA et al.; The role of indoor plantings as allergen sources was assessed by direct sampling of interior air . Homes with 10 or more plants in one room and three University of Michigan greenhouses were studied by means of a dc-powered rotorod and separate Andersen viable sampler collections incubated at 23 degrees and 50 degrees C . Sequential 30 and 60 sec Andersen samples were obtained during 15 min rotorod collections before and during watering of plants as well as during disturbance of foliage by a small fan . Relative humidity averaged 51% in homes and 78% in greenhouses . Aspergillus fumigatus recoveries were rare . Thermophiles, primarily bacteria, were present at low-to-moderate levels in homes, did not increase with watering of fan in homes, and rose only slightly with disturbance at greenhouse sites . Cladosporium and Penicillium dominated Andersen collections . Watering and fan increased levels of these taxa as well as rotorod recoveries of Alternaria . Epicoccum, and Pithomyces slightly in homes and markedly at greenhouse sites . We conclude that modest numbers of undisturbed house plants contribute minimally to aeroallergen prevalence in homes . However, especially under greenhouse conditions, plantings can harbor abundant fungus growth that may become airborne, especially when agitated directly.

Mol Cell Biol, 1982 Aug, 2(8), 930 - 8
Mutant strains of Tetrahymena thermophila defective in thymidine kinase activity: biochemical and genetic characterization; Cornish KV et al.; Three mutant strains, one conditional, of Tetrahymena thermophila were defective in thymidine phosphorylating activity in vivo and in thymidine kinase activity in vitro . Nucleoside phosphotransferase activity in mutant cell extracts approached wild-type levels, suggesting that thymidine kinase is responsible for most, if not all, thymidine phosphorylation in vivo . Thymidine kinase activity in extracts of the conditional mutant strain was deficient when the cells were grown or assayed or both at the permissive temperature, implying a structural enzyme defect . Analysis of the reaction products from in vitro assays with partially purified enzymes showed that phosphorylation by thymidine kinase and nucleoside phosphotransferase occurred at the 5' position . Genetic analyses showed that the mutant phenotype was recessive and that mutations in each of the three mutant strains did not complement, suggesting allelism.

Nucleic Acids Res, 1982 Jul 24, 10(14), 4409 - 12
The nucleotide sequences of 5S rRNAs from three ciliated protozoa; Kumazaki T et al.; The nucleotide sequences of 5S rRNAs from three ciliated protozoa, Paramecium tetraurelia, Tetrahymena thermophila and Blepharisma japonicum have been determined . All of them are 120 nucleotides long and the sequence of probable tRNA binding site of position 41-44 is GAAC which is characteristic of the plant 5S rRNAs . The sequence similarity percents are 87% (Paramecium/Tetrahymena), 86% (Paramecium/Blepharisma) and 79% (Tetrahymena/Blepharisma), suggesting a close relationship of these three ciliates.

Nucleic Acids Res, 1982 Jul 24, 10(14), 4279 - 91
Rearrangement of repeated DNA sequences during development of macronucleus in Tetrahymena thermophila; Iwamura Y et al.; Three clones of non-repetitive sequences and six clones containing repetitive sequences were obtained from micronuclear DNA of Tetrahymena thermophila . All the non-repetitive and three repetitive sequences had the same organization in micro- and macronuclear DNAs as revealed by blot hybridization . On the other hand, the remaining three clones with repetitive sequences had apparently different organization in the two nuclear DNAs . All these repetitive sequences showed a smear on the blot in addition to a number of discrete bands when micronuclear DNA was digested with EcoR I . In macronuclear DNAs, these sequences invariably became one or two bands and the smear disappeared . We conclude that, when a macronucleus develops from a micronucleus, the non-repetitive sequences amplify by more than 20 times with relatively few rearrangement, whereas some selected portions of repeated and/or repeat-contiguous sequences are amplified with rather extensive reorganization.

Biochim Biophys Acta, 1982 Jul 16, 717(1), 76 - 85
Purification and properties of galactokinase from Tetrahymena thermophila; Lavine JE et al.; Galactokinase (EC 2.7.1.6; ATP: D-galactose-1-phosphototransferase) was purified 152-fold with an 11% yield from Tetrahymena thermophila maximally derepressed for enzyme synthesis in late stationary phase . The purification procedure utilized sequential acid precipitation, batch DEAE-Sephacel chromatography, differential ammonium sulfate precipitation and narrow range electrofocusing . The apparent molecular weight of the holoenzyme as determined by gel filtration on Sephadex G-200 is 50000-55000 . The holoenzyme consists of two subunits of approx . 28000 daltons each, as determined by SDS-polyacrylamide gel electrophoresis . The native enzyme appears to be a single species with an isoelectric point at pH 5.1 . Optimal activity was obtained at pH 7.8 and 41 degrees C, with no added monovalent salt . D-Galactose, 2-deoxygalactose and galactosamine all are suitable carbohydrate substrates for the stereospecific galactokinase; only substitution at the C-2 position of galactose retains enzyme recognition . The enzyme utilizes ATP, 2'-dATP and 3'-dATP as phosphate donors; ADP and adenosine-5'-{gamma-thio}triphosphate are inhibitory . The Km values for galactose and ATP were determined to be 0.60 mM and 0.15 mM, respectively . The enzyme requires a divalent cation for activity, with effectiveness being in the order: Mg2+ greater than Co2+ greater than Mn2+ greater than Fe2+ . Galactokinases from all eucaryotic sources studied thus far seem to be very similar . Based upon the results reported here, the galactokinases from Tetrahymena and yeast appear to be most similar in their biophysical and biochemical properties.

J Biol Chem, 1982 Jul 10, 257(13), 7388 - 95
A thermostable tRNA (guanosine-2')-methyltransferase from Thermus thermophilus HB27 and the effect of ribose methylation on the conformational stability of tRNA; Kumagai I et al.; An S-adenosylmethionine-=dependent tRNA (guanosine-2'-)-methyltransferase (EC 2.1.1.34) was purified to the homogeneous state (2,400-fold) from a cell-free extract of an extreme thermophile, Thermus thermophilus HB27 . The enzyme was highly resistant to heat as reported for other enzymes from thermophilic organism . The enzyme is monomeric and its molecular weight was estimated to be about 20,000 . The Km values for S-adenosylmethionine and for Escherichia coli tRNAPhe were determined to be 0.47 microM and 10 nM, respectively, while the Ki for a competitive inhibitor S-adenosylhomocysteine, was 1.67 microM . When yeast tRNAPhe was methylated with the purified Gm-methyltransferase, a stoichiometric amount of methyl group was incorporated into the invariant guanosine at position 18 in the D-loop . Yeast tRNAPhe and E . coli tRNAMet, which were quantitatively methylated with the enzyme, were very similar to the native tRNAs with regard to amino acid acceptor activity and melting temperature, but were more resistant to RNase T1 and RNase A digestions than the corresponding native tRNAs.

Biochemistry, 1982 Jul 6, 21(14), 3294 - 8
Complete amino acid sequence of the 4Fe-4S, thermostable ferredoxin from Clostridium thermoaceticum; Elliott JI et al.; The complete amino acid sequence of the 4Fe-4S ferredoxin from the thermophilic bacterium Clostridium thermoaceticum has been determined . The protein is extremely thermostable and is the only known clostridial ferredoxin to contain a single {4Fe-4S} cluster . The sequence totals 63 residues and includes the first tryptophan (Trp-26) reported for a clostridial ferredoxin, and other amino acids not commonly found in clostridial or clostridial-like ferredoxins: methionine (Met-1), histidine (His-33), arginine (Arg-49), and leucine (Leu-9, -19, and -31) . Sequence homology to clostridial and other 8Fe-8S ferredoxins is limited to eight to nine residues at the amino-terminal sulfhydryl grouping (Cys-10, -13, -16, and -20) and two to five residues in the carboxyterminal region . This ferredoxin is, thus, sequentially distinct from all known clostridial ferredoxins and from other bacterial ferredoxins in both the 8Fe-8S and 4Fe-4S classes.

Mikrobiologiia, 1982 Jul-Aug, 51(4), 611 - 5
{Cytological characteristics of the thermophilic bacterium Thermus ruber}; Shadrina IA et al.; The obligate thermophilic bacterium Thermus ruber 12b was examined using optical and electron microscopy . Its cells were shown to be polymorphous . In a liquid medium, the cells associate yielding complex spherical bodies having a surface layer in common and complexes consisting of two or more cells but without a common envelope . A large nucleoid is located in the central part of the cells; it contains polyphosphate granules . Fat inclusions and analogues of mitochondria with dehydrogenase activity typical of them can be detected at the periphery . The cell wall consists of a folded outer membrane, an underlying fibrillar material, and a thin rigid layer . Peculiar adhesion zones appear between the outer membrane and the rigid layer in places where folds are formed . An electron-dense substance is deposited on the surface of the three-layer cytoplasmic membrane.

J Allergy Clin Immunol, 1982 Jul, 70(1), 11 - 4
Evaluation of the patient for occupational immunologic lung disease; Fink JN; Careful evaluation of the worker patient is essential to detect occupational immunologic disease (OILD) and to define the source of the illness . The history is essential, especially in view of the temporal relationships between symptoms and exposure: the presence of variable latent periods between exposure and symptoms must be considered . Once OILD is suspected, additional evaluation is of value . Physical examination may be normal or may detect features of asthma or interstitial disease, depending on the stage of the illness . Chest x-ray evaluation may also reflect the type and stage of illness . Serologic studies may detect precipitating antibodies against environmental antigens . Skin tests with common antigens are useful in detecting an atopic background . Environmental studies may reveal particular antigens in the form of fungi, thermophilic actinomycetes, or chemicals that are volatile, soluble, or could be aerosolized . Pulmonary function studies before and after the work shift or after a period of avoidance may provide clues as to the environmental nature of the illness . Bronchopulmonary challenge with occupational environmental materials may be valuable in obtaining a definitive diagnosis . Therapy of OILD depends on the worker's response, degree of illness, and feasibility of avoidance of protection.

Biochem J, 1982 Jul 1, 205(1), 147 - 52
Purification and partial characterization of a thiol proteinase from the thermophilic fungus Humicola lanuginosa; Shenolikar S et al.; An extracellular thiol proteinase was produced by the growth of a thermophilic fungus, Humicola lanuginosa, on a medium containing 2% casein, and was purified to virtual homogeneity by affinity chromatography on organomercurial columns . The essential thiol group for activity was confirmed by the inhibition of the enzyme by p-chloromercuribenzoate and mercuric ions . The enzyme, purified 27-fold from the extracellular fluid, exhibited an Mr of 23700 on gel filtration and sedimentation equilibrium . The H . lanuginosa proteinase preferentially cleaves at the C-terminal end of hydrophobic amino acid residues . This proteinase differed from the plant enzyme papain in its interaction with three affinity matrices and its substrate specificity towards synthetic substrates . This enzyme represents a unique example of a thiol proteinase obtained from a fungal source.

J Bacteriol, 1982 Jul, 151(1), 328 - 33
Isolation and characterization of an Fe,-S8 ferredoxin (ferredoxin II) from Clostridium thermoaceticum; Elliott JI et al.; A second ferredoxin protein was isolated from the thermophilic anaerobic bacterium Clostridium thermoaceticum and termed ferredoxin II . This ferredoxin was found to contain 7.9 +/- 0.3 iron atoms and 7.4 +/- 0.4 acid-labile sulfur atoms per mol of protein . Extrusion studies of the iron-sulfur centers showed the presence of two {Fe4-S4} centers per mol of protein and accounted for all of the iron present . The absorption spectrum was characterized by maxima at 390 nm (epsilon 390 = 30,400 M-1cm-1) and 280 nm (epsilon 280 = 41.400 M-1 cm-1) and by a shoulder at 300 nm . The ration of the absorbance of the pure protein at 390 nm to the absorbance at 280 nm was 0.74 . Electron paramagnetic resonance data showed a weak signal in the oxidized state, and the reduced ferredoxin exhibited a spectrum typical of {Fe4-S4} clusters . Double integration of the reduced spectra showed that two electrons were necessary for the complete reduction of ferredoxin II . Amino histidine, and 1 arginine, and a molecular weight of 6,748 for the native protein . The ferredoxin is stable under anaerobic conditions for 60 min at 70 degrees C . The average oxidation-reduction potential for the two {Fe4-S4} centers was measured as -365 mV.

Eur J Biochem, 1982 Jun 15, 125(1), 143 - 9
On the properties of immobilized elongation factor Tu from Thermus thermophilus HB8; Fischer W et al.; Elongation factor Tu from Thermus thermophilus was coupled to cyanogen-bromide-activated Sepharose 4B and its properties were investigated . The immobilized elongation factor retained its ligand binding properties . It specifically binds GDP and GTP but does not interact with other nucleotide 5'-phosphates . A conversion of immobilized EF-Tu . GDP to EF-Tu . GTP can be achieved by simple equilibration with GTP . The immobilized EF-Tu . GTP specifically binds aminoacyl-tRNAs and allows a facile purification of aminoacyl-tRNA species from bulk tRNA . It is stable at room temperature for several months and can be repeatedly used for aminoacyl-tRNA isolation.

J Embryol Exp Morphol, 1982 Jun, 69, 83 - 105
Regulation of the pattern of basal bodies within the oral apparatus of Tetrahymena thermophila; Bakowska J et al.; The number and arrangement of basal bodies included in the four compound ciliary organelles making up the mature oral apparatus of Tetrahymena thermophila ordinarily vary only slightly . Severe starvation brings about formation of oral structures with a reduced number of basal bodies within these organelles, and sometimes with a complete loss of one of the component organelles . Such reductions are stringently specified in spatial terms, but they do not represent simple and proportional shrinkage of the organelle complex . Instead, certain spatial features remain essentially unaltered, while others undergo major quantitative reductions, resulting in large changes in the internal proportions of the structures . This selective regulation can be explained in terms of the different parallel and sequential processes taking place during the development of this organelle complex . There is also no strict proportionality between the size of the oral apparatus and that of the cell; instead, oral apparatuses become relatively larger as cells become smaller . This is due in part to the inherent temporal discontinuity of oral development, but there is probably also a real change in the oral/body size relation at the time of oral development . The 'French flag' rule fails when applied to the relative sizes and internal proportions of organelle systems in this and in other ciliates.

Biochem J, 1982 Jun 1, 203(3), 787 - 90
The purification and some properties of a stereospecific D-asparaginase from an extremely thermophilic bacterium, Thermus aquaticus; Guy GR et al.; A specific D-asparaginase was isolated and crystallized from Thermus aquaticus strain T351 . It is present in larger amounts than the L-asparaginase . The enzyme has a molecular weight of 60 000, an isoelectric point of 4.8 and a Km of 2 mM . It has 6 disulphide bonds/molecule, and a histidine residue at the active site . It is inhibited by keto acids and by high salt concentrations.

Appl Environ Microbiol, 1982 Jun, 43(6), 1343 - 53
Recovery of Campylobacter jejuni and Campylobacter coli from inoculated foods by selective enrichment; Doyle MP et al.; A direct enrichment procedure was developed to selectively recover small numbers of Campylobacter jejuni, C . coli, and nalidixic acid-resistant thermophilic Campylobacter from foods . The procedure includes an enrichment medium composed of brucella broth, 7% lysed horse blood, 0.3% sodium succinate, 0.01% cysteine hydrochloride, vancomycin (15 micrograms/ml), trimethoprim (5 micrograms/ml), polymyxin B (20 IU/ml), and cycloheximide (50 micrograms/ml) that is inoculated with 10 or 25 g of food and incubated with agitation under microaerophilic conditions at 42 degrees C for 16 to 18 h . After incubation, the medium is plated directly onto Campy-BAP agar plates (M . J . Blaser et al., Ann . Intern . Med . 91:179-185, 1979), and resulting colonies that resemble Campylobacter are identified by conventional tests . The foods evaluated included raw milk, hamburger, and chicken skin which had aerobic plate counts of 10(5) to 10(9) bacteria/g . The procedure was effective in recovering as few as 0.1 cell of Campylobacter per g of food . Of the 50 isolates of Campylobacter evaluated, all were recovered from raw milk and hamburger at a level of 1 to 4 cells/g, and 41 and 40 isolaes were recovered from the hamburger and milk, respectively, at 0.1 to 0.4 cell/g . The enrichment was least effective for recovering campylobacters from chicken skin, as 7 and 26 of 50 isolates were not recovered at 1 to 4 and 0.1 to 0.4 cell/g, respectively . This new procedure is more rapid, direct, and effective than other enrichment or direct plating procedures for recovering small numbers of campylobacters from foods.

J Bacteriol, 1982 Jun, 150(3), 1391 - 9
Differential metabolism of cellobiose and glucose by Clostridium thermocellum and Clostridium thermohydrosulfuricum; Ng TK et al.; Clostridium thermohydrosulfuricum consumed glucose in preference to cellobiose as an energy source for growth . The rates of substrate uptake in glucose- and cellobiose-grown cell suspensions were 45 and 24 nmol/min per mg (dry weight), respectively, at 65 degrees C . The molar growth yields (i.e., grams of cells per mole of glucose equivalents) were similar on cellobiose and glucose (19 and 16, respectively) . Both glucose- and cellobiose-grown cells contained a glucose permease activity and high levels of hexokinase (greater 0.34 mumol/min per mg of protein at 40 degrees C) . Growth on cellobiose was associated with induction of a cellobiose permease activity . In contrast, Clostridium thermocellum metabolized cellobiose in preference to glucose as an energy source and displayed lower growth rates on both substrates . The substrate uptake rates in cellobiose- and glucose-grown cell suspensions were 18 and 17 nmol/min per mg (dry weight), respectively . The molar yields were 38 on cellobiose and 20 on glucose . Extracts of glucose- and cellobiose-grown cells both contained cellobiose phosphorylase and phosphoglucomutase activities, whereas only glucose-grown cells contained detectable levels of glucose permease and hexokinase activities . The general catalytic and kinetic properties of the glucose- and cellobiose-catabolizing enzymes in the two species are described, and a model is proposed to distinguish differential saccharide metabolism by these thermophilic ethanologens.

Nucleic Acids Res, 1982 May 11, 10(9), 2823 - 38
The intervening sequence excised from the ribosomal RNA precursor of Tetrahymena contains a 5-terminal guanosine residue not encoded by the DNA; Zaug AJ et al.; The ribosomal RNA precursor of Tetrahymena thermophila contains a 0.4 kilobase intervening sequence that is excised as a linear RNA molecule ("IVS RNA") and subsequently cyclized . In vitro transcription in isolated nuclei was used to accumulate the IVS RNA labeled at its 5' end was subjected to sequencing gel analysis and terminal nucleotide analysis . In addition, uniformly labeled IVS RNA was cleaved with RNAase T1, and the resulting oligonucleotides were studied by two-dimensional fingerprinting . The IVS RNA was found to be a unique molecule with no discernible terminal heterogeneity . The 5'-terminal nucleotide is a guanosine that is not present at the corresponding point in the DNA sequence, determined by N . Kan and J . Gall (see adjoining paper) . This nucleotide is added to the IVS during splicing {Cech, Zaug, and Grabowski (1981) Cell 27, 487-496} . Based on the sequences near the ends of the RNA, the remainder of the RNA sequence is colinear with that of the DNA . The IVS RNA has 5'-monophosphate and 3'-hydroxyl termini . Comparison of these results to those obtained previously for yeast tRNA intervening sequences leads us to conclude that the splicing mechanisms are fundamentally different for these two classes of transcripts.

Nucleic Acids Res, 1982 May 11, 10(9), 2809 - 22
The intervening sequence of the ribosomal RNA gene is highly conserved between two Tetrahymena species; Kan NC et al.; The entire intervening sequence of Tetrahymena thermophila ribosomal DNA has been determined . It is 413 nucleotides long and has the same splice junctions as those in T . pigmentosa . There is 93% homology between the intervening sequences in the two species, and 100% homology between their adjacent 26S RNA coding regions.

J Bacteriol, 1982 May, 150(2), 993 - 6
Sequence specificity of DNA adenine methylase in the protozoan Tetrahymena thermophila; Bromberg S et al.; The sequence specificity of the Tetrahymena DNA-adenine methylase was determined by nearest-neighbor analyses of in vivo and in vitro methylated DNA . In vivo all four common bases were found to the 5' side of N6-methyladenine, but only thymidine was 3' . Homologous DNA already methylated in vivo and heterologous Micrococcus luteus DNA were methylated in vitro by a partially purified DNA-adenine methylase activity isolated from Tetrahymena macronuclei . The in vitro-methylated sequence differed from the in vivo sequence in that both thymidine and cytosine were 3' nearest neighbors of N6-methyladenine.

J Med Microbiol, 1982 May, 15(2), 247 - 51
Sensitivity of thermophilic campylobacters to R-type pyocines of Pseudomonas aeruginosa; Blackwell CC et al.; Strains of thermophilic campylobacters of human origin were examined for bacteriocine activity and for susceptibility to R-type pyocines of Pseudomonas aeruginosa . None of 50 strains inhibited the growth of any other strain, but 13 of 80 strains (16%) were sensitive to R-type pyocines . Absorption of one of the partially purified pyocine preparations with a sensitive strain resulted in the removal of pyocine activity and a decrease in viable count of the organism by 85%.

J Protozool, 1982 May, 29(2), 251 - 8
Electrophoretic analysis of endonuclease-generated fragments of k-DNA, of esterase isoenzymes, and of surface proteins as aids for species identification of insect trypanosomatids; Camargo EP et al.; In order to verify the applicability of biochemical methods for species identification of Trypanosomatidae, 13 species of monoxenic trypanosomatids plus the heteroxenous Trypanosoma cruzi were comparatively analyzed by three different biochemical methods . Insect trypanosomatids examined were: Crithidia acanthocephali, C . fasciculata (three varieties), C . luciliae luciliae, C . luciliae thermophila, C . deanei, C . oncopelti, Herpetomonas muscarum muscarum, H . megaseliae, H . samuelpessoai, H . mariadeanei, Leptomonas seymouri, L . collosoma, L . samueli, and Blastocrithidia culicis . Also included in the survey were aposymbiotic strains of C . deanei and C . oncopelti . Methods used were: electrophoretic profiling of endonuclease-generated fragments of k-DNA, esterase isoenzymes profiling, and polyacrylamide-gel electrophoresis (SDS-PAGE) of radioiodinated cell surface proteins . Interspecific but not intraspecific differences were detected by all three methods among the 13 monoxenic species examined . Thus, it is concluded that these methods can be successfully used, in addition to classical criteria, for species identification of insect trypanosomatids.

Biochem J, 1982 Apr 1, 203(1), 277 - 84
Characterization of a cellobiose dehydrogenase in the cellulolytic fungus Sporotrichum (Chrysosporium) thermophile; Coudray MR et al.; An extracellular enzyme from culture filtrates of Sporotrichum (Chrysosporium) thermophile (A.T.C.C . 42 464) after growth on cellulose or cellobiose was shown to oxidize cellobiose to cellobionic acid in vitro . Lactose and cellodextrins were also efficiently oxidized, but the enzyme was not active against most mono- and di-saccharides . Several redox substances could act as electron acceptors, but molecular oxygen, tetrazolium salts and NAD(P) were not reduced . Activity was stimulated up to 2-fold in the presence of 0.05 M-Mg2+ . The pH optimum of the enzymic reaction was acidic when the activity was tested with dichlorophenol-indophenol or Methylene Blue, but was neutral to alkaline for 3,5-di-t-butyl-1,2-benzoquinone or phenazine methosulphate as electron acceptors . As the enzyme was formed inductively in parallel with the endocellulase, its possible function in relation to cellulolysis is discussed.

J Biochem (Tokyo), 1982 Apr, 91(4), 1343 - 8
Heat-stable and fructose 1,6-bisphosphate-activated L-lactate dehydrogenase from an extremely thermophilic bacterium; Taguchi H et al.; Heat-stable L-lactate dehydrogenase {EC 1.1.1.27} was purified from an extremely thermophilic bacterium belonging to the genus Thermus, and it showed an allosteric nature dependent on fructose 1,6-bisphosphate as an effector . The enzyme had a molecular weight of approximately 120,000 with a subunit molecular weight of 31,000 . For pyruvate reduction, the optimal pH was found to be 4.5 . At neutral pH, which is a more physiological region, little enzyme activity was observed, but marked reaction resulted from the addition of fructose 1,6-bisphosphate . This addition stabilized the enzyme toward heat treatment at up to 95 degrees C . The optimal temperature for the enzyme reaction was approximately 80 degrees C for pyruvate reduction and 95 degrees C for lactate oxidation.

J Cell Sci, 1982 Apr, 54, 137 - 47
Age-associated change in macronuclear DNA content in Paramecium caudatum; Takagi Y et al.; Macronuclear DNA content in Paramecium caudatum was found to be almost unchanged with a mean of about 400C during the earlier two-thirds of the life span in terms of the number of fissions and then it dropped rapidly to about one-fifth of the initial content . The age when rapid DNA decline occurred corresponded to that when the characteristics of senescence appeared . This decreasing pattern of macronuclear DNA content contrasted with earlier observations in P . tetraurelia, P . bursaria and Tetrahymena thermophila . The data suggested that in P . caudatum the distribution pattern of macronuclear DNA to daughter cells changed from an almost equal distribution in younger cells to an unequal distribution in older cells, while the relative volume of the macronucleus to the whole cell remained almost constant throughout the life cycle.

Clin Chem, 1982 Apr, 28(4 Pt 2), 993 - 7
Characterization of allergen extracts by two-dimensional electrophoretic techniques: Micropolyspora faeni antigens; Vesterberg O et al.; Thermophilic actinomycetes and saprobic fungi are important in the etiology of allergic occupational diseases such as "farmer's lung" disease . Each such organism produces several protein antigens . Inhaled, these antigens stimulate production of antibodies . Detection of precipitating antibodies has been useful in the diagnosis of diseases so induced . Characterization of allergen extracts from microorganisms associated with these diseases is important, to improve the sensitivity and precision of the precipitin analysis . For this purpose we submitted crude allergen extracts to electrophoresis and isoelectric focusing in agarose gels . Staining the gels revealed many protein components in each extract, especially after isoelectric focusing . After separation in one dimension, a lane of gel was cut out and the proteins were electrophoresed at right angles into another gel, which contained antibodies . Several arcs of immunoprecipitates, indicating different antigens, were seen . This technique ("crossed immunoelectrofocusing") has earlier been used with polyacrylamide in the first dimension, but it is improved by using instead agarose of a special quality . Further to improve the quantification, we isolated pieces of gel containing the proteins of interest and used them as samples in zone immunoelectrophoresis assay . This method is straightforward, easy to evaluate, and about 100-fold as sensitive as radial immunodiffusion . The amount of protein in each sample is usually proportional to the distance from the upper gel surface to the front of each immunoprecipitate . The increased sensitivity allows study of many hitherto unexamined antigens.

J Biochem Biophys Methods, 1982 Apr, 6(1), 31 - 7
A modification for increasing the sensitivity of the casein-agar plate assay: a simple semiquantitative assay for thermophilic and mesophilic proteases; Cowan DA et al.; A casein-agar plate assay was used for the quantitative determination of both mesophilic and thermophilic proteases . Because many proteases are thermostable, assay at higher temperatures is possible . The sensitivity of the plate assay increased with temperature, the optimum assay temperature depending on the thermostability of the enzyme (e.g . Thermus protease, 75 degrees C; thermolysin, 65 degrees C; trypsin, 65 degrees C; alpha-chymotrypsin, 45 degrees C) . A positive correlation was observed between incubation temperature and the density of the para-casein precipitate, increasing the accuracy of diameter measurement . Using this modified method, thermostable proteases could be assayed at levels well below the limits of detection of other methods (e.g . 40 pg of thermolysin and 300 pg of trypsin detectable at 65 degrees C, a 16-fold increase in the sensitivity for trypsin compared with a conventional plate assay (Fossum, K . (1970) Acta Pathol . Microbiol . Scand . Sect . B 78, 350-361)) . The sensitivity of the plate assay could be further increased by the inclusion of some detergents and chaotropic agents in the gel.

Mol Cell Biol, 1982 Apr, 2(4), 378 - 85
Selection and characterization of a glucokinase-deficient mutant of Tetrahymena thermophila; Roberts CT Jr et al.; We have isolated a mutant of Tetrahymena thermophila that is resistant to inhibition of growth by the glucose analog 2-deoxyglucose . The mutant exhibits a deficiency in a cytoplasmic glucokinase . This enzymatic defect and the attendant inability to convert 2-deoxyglucose to toxic phosphorylated derivatives is apparently the sole basis for the mutant phenotype since transport of glucose and 2-deoxyglucose is unimpaired; there is no elevation of glucose-6-phosphatase activity, which could decrease the level of toxic 2-deoxyglucose metabolites . Genetic analyses have shown that the mutant allele is recessive and inherited as a single Mendelian mutation . The glucokinase-deficient strain described here is useful for the selection of other mutants in this organism and for the investigation of various cellular processes initiated or modulated by glucose and its analogs . We have exploited the molecular defect in this strain to investigate the initial steps in the cyclic AMP-mediated repression of galactokinase gene expression which is caused by glucose.

Nucleic Acids Res, 1982 Mar 25, 10(6), 2145 - 61
Regulation of protein synthesis in Tetrahymena: isolation and characterization of polysomes by gel filtration and precipitation at pH 5.3; Calzone FJ et al.; The fraction of ribosomes loaded on polysomes is about 95% in logarithmically growing Tetrahymena thermophila, and about 4% in starved cells . Cytoplasmic extracts from cells in these two physiological states were used to develop column chromatographic methods for the purification of polysomes . Bio-Gel A 1.5 m was found to separate total cytoplasmic ribosomes from many soluble proteins, including RNAse, with no detectable change in the polysome size distribution . Polysomes can be separated from monosomes and non-polysomal mRNA by chromatography on Bio-Gel A 15 m without size selection . These methods can easily be adapted to large scale preparations of polysomes, even from cells where a small fraction of the ribosomes is on polysomes . A method is described for reversible precipitation of polysomes and monosomes from dilute solutions at pH 5.3 which greatly facilitates polysome isolation . Hybridization of 3H-labeled polyU to RNA isolated from column fractions has been used to demonstrate that purification of EDTA released polysomal mRNA can be performed using the column chromatography procedures described here . These methods have been employed to demonstrate that most of the cytoplasmic mRNA in log-phase Tetrahymena is loaded onto polysomes while most of the mRNA is starved cells exists in a non-polysomal form.

Nucleic Acids Res, 1982 Mar 11, 10(5), 1503 - 13
The nucleotide sequence at the transcription termination site of the ribosomal RNA gene in Tetrahymena thermophila; Din N et al.; The sequence of 415 nucleotides surrounding the transcription termination site for ribosomal RNA in Tetrahymena thermophila has been determined . The positions of the 3'-ends of mature 26S rRNA, pre-26S rRNA and 35S pre-rRNA were localized within this sequence by hybridization of the purified RNA species to be selected DNA fragments, followed by S1 nuclease treatment of the hybrid and a precise sizing of the RNA-protected DNA fragments on sequencing gels . The 35S pre-rRNA population contained molecules with two distinct 3'-ends, one of which is identical to the end of pre-26S and 26S rRNA, while the other corresponds to a position 15 nucleotides further downstream, which is assumed to be the transcription termination site . The non-coding DNA strand contains a cluster of T's at the putative termination site, and several other T clusters are found further downstream . A short inverted repeat sequence is located near the putative termination site within the transcribed region . The possible role of these structures for transcription termination is discussed.

Biochim Biophys Acta, 1982 Mar 8, 685(3), 283 - 8
Liposomal membranes . XI . A suggestion to structural characteristics of acido-thermophilic bacterial membranes; Sunamoto J et al.; To understand the role of omega-cyclohexyl fatty acid residue of lipids in acido-thermophilic bacterial membranes, three unusual phosphatidylcholines, 1, 2-di-11-cyclohexylundecanoyl-L-alpha-phosphatidylcholine (11CYPC), 1,2-di-13-cyclohexyltridecanoyl-L-alpha-phosphatidylcholine (13CYPc), and 1-13-cyclohexyltridecanoyl-2-11-cyclohexylundecanoyl-L-alpha- phosphatidylcholine (1-13CY-2-11CYPC) were prepared and the steady-state fluorescence anisotropy of 1, 6-diphenylhexatriene (DPH) in the hydrophobic domain of these liposomal bilayers was determined . Compared with the case of dipalmitoyl (DPPC) or dimyristoyl phosphatidylcholine (DMPC), introducing the omega-cyclohexyl moiety onto lecithins makes the bilayers fluid below the phase transition temperature, while immobilizes them above the phase transition temperatures . The properties of the unusual phosphatidylcholine liposomes suggested by the steady-state fluorescence anisotropy investigation were in good agreement with those obtained from the thermotropic and permeability investigations . Results obtained are discussed from the view point of the role and function of lipid membranes of acido-thermophilic bacteria which contain unusual fatty acids.

Mikrobiologiia, 1982 Mar-Apr, 51(2), 296 - 301
{Carboxysomes of the thermophilic hydrogen bacterium Pseudomonas thermophila}; Romanova AK et al.; The technique of electron microscopy was used to detect the presence of paracrystal hexagonal inclusions in the cells and spheroplasts of the thermophilic hydrogen bacterium Pseudomonas thermophila . The connection of the inclusions with DNA threads can easily be seen on photomicrographs of the spheroplasts . The cells were disintegrated by freezing and thawing and the resultant homogenates were centrifuged in a sucrose density gradient; up to 80% of the activity of ribulose-1,5-diphosphate carboxylase (RDP carboxylase, EC 4.1.1.39) was found in the fraction of particles containing hexagonal paracrystal inclusions (carboxysomes) . Their granular content and the tendency to diffuse were seen at a magnification x100,000 . The percentage of the insoluble enzyme was higher in cells in the stationary growth phase than in growing cells . Only 25% of the enzyme activity was detected in the particles after the cells treated with lysozyme had been subjected to osmotic shock . A possible role of carboxysomes in cells as a compartment storing RDP carboxylase is discussed.

Appl Environ Microbiol, 1982 Mar, 43(3), 561 - 5
Response of Campylobacter jejuni to sodium chloride; Doyle MP et al.; Studies were done to provide more comprehensive information on the response of Campylobacter jejuni and nalidixic acid-resistant, thermophilic Campylobacter (NARTC) to sodium chloride at 4, 25, and 42 degrees C . Three strains of C . jejuni were studies, and all could grow at 42 degrees C in the presence of 1.5% NaCl, but not 2.0% NaCl . At the same temperature, NARTC could grow in 2.0% NaCl and was substantially more tolerant to 2.5 and 4.5% NaCl than was C . jejuni . Both C . jejuni and NARTC grew poorly in the absence of added NaCl and grew best in the presence of 0.5% NaCl at 42 degrees C . At 25 degrees C, NaCl concentrations of 1.0 to 2.5% were protective to NARTC, but the same concentrations of salt generally enhanced the rate of death of C . jejuni . At 4 degrees C, both C . jejuni and NARTC were sensitive to 1.0% or more NaCl; however, the rate of death at this temperature was substantially less than that which occurred at 25 degrees C . A 3 log10 decrease of cells occurred in 4.5% NaCl after 1.2 to 2.1 days at 25 degrees C, and a similar reduction in cells took approximately 2 weeks at the same salt concentration and 4 degrees C . Although C . jejuni grows best in the presence of 0.5% NaCl, the presence of NaCl at concentrations as low as 1.0% may retard growth or increase rate of death; hence, it is advisable that growth media used for recovering or enumerating this organism contain 0.5% NaCl, but not 1.0% or more NaCl.

Eur J Biochem, 1982 Mar 1, 122(3), 471 - 7
DNA-dependent RNA polymerase of thermoacidophilic archaebacteria; Prangishvilli D et al.; The component compositions of the DNA-dependent RNA polymerases of the extremely thermophilic, anaerobic sulfur-respiring archaebacteria Thermoproteus tenax and Desulfurococcus mucosus strongly resemble each other but also that of the RNA polymerase of Sulfolobus acidocaldarius suggesting that both organisms belong to the same novel order Thermoproteales, which together with the order represented by Sulfolobus, forms the thermoacidophilic branch of archaebacteria . The component pattern of the RNA polymerase of Thermoplasma acidophilum, which does not belong to this branch, also appears homologous . The archaebacterial type of the DNA-dependent RNA polymerase is thus characterized by 9-10 components yielding a characteristic pattern which resembles that of yeast RNA polymerase A(I) . In contrast to the alpha subunit of eubacterial RNA polymerases, the third largest component of archaebacterial RNA polymerases, although similar in size, is present only one per enzyme monomer . The polymerases of T . tenax and D . mucosus, like those previously isolated from other archaebacteria, are completely resistant against 100 microgram/ml rifampicin and streptolydigin . The RNA polymerases of both organisms are highly thermostable . The enzyme from D . mucosus transcribes selectively and almost completely the H strand of phase T7 DNA.

Can J Biochem, 1982 Mar, 60(3), 398 - 407
DNA binding proteins from Tetrahymena thermophila; Tsao NN et al.; We have used DNA-cellulose chromatography to isolate single-strand binding proteins from Tetrahymena thermophila . Three major proteins which bind to denatured DNA-cellulose were obtained . The predominant protein has a molecular weight of 20 000 in sodium dodecyl sulfate - polyacrylamide gel electrophoresis and possesses many of the properties of the helix destabilizing proteins isolated from prokaryotic and eukaryotic sources . The protein facilitates denaturation of the synthetic copolymer poly{d(A-T).d(A-T)}, depressing the melting temperature by nearly 40 degrees C . It also permits the renaturation of poly{d(A-T)}.d(A-T)} in high salt concentration . Two other binding proteins have molecular weight of 25 000 and 23 000 in sodium dodecyl sulfate - polyacrylamide gel electrophoresis . The protein with a molecular weight of 25 000 is probably the "M protein" previously isolated from Tetrahymena thermophila which has been shown to stimulate Tetrahymena DNA polymerase . These two proteins failed to show helix destabilizing, DNA dependent ATPase, or deoxyribonuclease activities . These three proteins are abundant in the cell with approximately 1.0 x 10(6) to 10.0 x 10(6) molecules of each protein monomer per cell . One molecule of each protein monomer binds to 7 to 10 nucleotides as detected by a nitrocellulose filter binding assay . Peptide mapping of the three proteins suggests that they are all distinct . We have also found that the binding proteins can interact with Tetrahymena DNA polymerase and some other proteins to form an enzyme complex, a putative replication complex.

Cell, 1982 Mar, 28(3), 595 - 604
Allele-specific, selective amplification of a ribosomal RNA gene in Tetrahymena thermophila; Pan WC et al.; The amplification of ribosomal DNA during development of the somatic macronucleus in Tetrahymena thermophila was analyzed by genetic and molecular biological techniques . We have identified an alternate form of the rDNA, structurally distinguishable from the wild-type by an extra cutting site for Bam HI in its nontranscribed spacer . The altered rDNA was inherited in crosses in a simple Mendelian fashion, consistent with the presence of only one rRNA gene copy per haploid genome in the micronucleus . We therefore define a locus for the rRNA structural gene, the rdnA locus, with the allele determining the alternate form designated rdnA1 . In over 95% of T . thermophila clones heterozygous for the rdnA locus in the micronucleus (rdnA1/rdn+), the macronucleus, which develops from a division product of this micronucleus, contained almost exclusively rdnA1-type amplified palindromic rDNA molecules . The rdnA1 allele is thus almost always dominant over the rdn+ allele with respect to amplification . This genetic variant of the rdnA locus was used to show that the single, free, nonpalindromic rRNA genes, which are synthesized during rDNA amplification, are derived from micronuclear gene copies from both chromosomal homologs . We therefore conclude that in these heterozygotes, selective amplification of the rdnA1 allele is not caused by the production of only one type of free, single rRNA gene during amplification.

J Bacteriol, 1982 Mar, 149(3), 824 - 30
Transformation of Bacillus stearothermophilus with plasmid DNA and characterization of shuttle vector plasmids between Bacillus stearothermophilus and Bacillus subtilis; Imanaka T et al.; A thermophilic bacterium Bacillus stearothermophilus IFO 12550 (ATCC 12980) was transformed with each of the following plasmids, pUB110 (kanamycin resistance, Kmr), pTB19 (Kmr and tetracycline resistance {Tcr}), and its derivative pTB90 (Kmr Tcr), by the protoplast procedure in the presence of polyethylene glycol at 48 degrees C . The transformation frequencies per regenerant for pUB110, pTB19, and pTB90 were 5.9 x 10(-3), 5.5 x 10(-3), and 2.0 x 10(-1), respectively . Among these plasmids, pTB90 was newly derived, and the restriction endonuclease cleavage map was constructed . When tetracycline (5 micrograms/ml) was added into the culture medium, the copy number of pTB90 in B . stearothermophilus was about fourfold higher than that when kanamycin (5 micrograms/ml) was added instead of tetracycline . Bacillus subtilis could also be transformed with the plasmids extracted from B . stearothermophilus and vice versa . Accordingly, pUB110, pTB19, and pTB90 served as shuttle vectors between B . stearothermophilus and B . subtilis . The requirements for replication of pTB19 in B . subtilis and B . stearothermophilus appear to be different, because some deletion plasmids (pTB51, pTB52, and pTB53) derived from pTB19 could replicate only in B . subtilis, whereas another deletion plasmid pTB92 could replicate solely in B . stearothermophilus . Plasmids pTB19 and pTB90 could be maintained and expressed in B . stearothermophilus up to 65 degrees C, whereas the expression of pUB110 in the same strain was up to 55 degrees C.

Ann Microbiol (Paris), 1982 Mar-Apr, 133(2), 319 - 42
Isoenzyme patterns of pathogenic and non-pathogenic Naegleria spp . using agarose isoelectric focusing; De Jonckheere JF; Using agarose isoelectric focusing, the isoenzyme patterns of 7 different enzymes were compared in 52 Naegleria strains . The pathogenic N . fowleri was found the most homogeneous species . N . lovaniensis seems to be constituted of different types which form nevertheless a cohesive group . Within N . gruberi, large interstrain band variations were found in almost all enzyme systems . A re-examination of the taxonomic position of this species may therefore be taken into consideration . High temperature strains from Australia were confirmed to be different from N . lovaniensis . Members of a new pathogenic Naegleria sp., N . australiensis, seem to occur in Europe . Large thermophilic strains with many large pores in the cysts show identical zymograms and may constitute a new species or genus.

Biophys J, 1982 Feb, 37(2), 563 - 6
Artificial black membranes from bipolar lipids of thermophilic Archaebacteria; Gliozzi A et al.; The membrane of thermophilic archaebacteria is characterized by the presence of unusual isoprenoid bipolar lipids . The molecular organization of these lipids is still a matter of study . Important information could come from forming artificial black membranes . Black films can be formed from n-alkane or squalene dispersions of bipolar lipids extracted from the membrane of Caldariella acidophila . Membrane formation occurred only above a critical temperature (approximately 70 degrees C) corresponding to the physiological one . At lower temperatures, special solvent systems (n-alkanes or squalene, butanol and n-alkanes or squalene, butanol chloroform) were required . To characterize the physical parameters of these membranes, conductance and capacitance measurements were performed . Conductance was in the range of 10(-8) - 10(-7) omega -1 cm -2 , where specific capacitance at T = 72 degrees C was Cs = 0.685 +/- 0.004 microF/cm2 and Cs = 0.658 +/- 0.08 microF/cm2, corresponding to a dielectric thickness of 27 and 29 A for squalene and dodecane dispersions, respectively . Capacitance was shown to vary as the square of membrane potential, as usual in lipid bilayers . Values of the proportionality constant alpha have been compared to those of solvent-containing and solvent-free bilayers . The behavior of capacitance as a function of temperature is also shown by lowering temperature; the occurrence of complex structural changes was indicated . All the experimental data suggest that the presence of solvent is very low . Two possible molecular configurations of the films are discussed.

Nucleic Acids Symp Ser, 1982, (11), 173 - 6
Single crystals of 5S rRNA from Thermus thermophilus HB8 and preliminary X-ray diffraction data; Morikawa K et al.; 5S rRNA from extremely thermophilic bacteria, Thermus thermophilus HB8, was crystallized . Using a vapour diffusion method, single crystals were grown with sufficient sizes for x-ray diffraction study . The crystals belong to space group P3(1)21 (or enatiomorph P3(2)21) with a = 99 A, b = 99 A and c = 360 A . A unit cell contains probably 36 molecules corresponding to 6 molecules/asymmetric unit . A smaller pseudo-lattice seems to be constructed with the same lengths for a- and b-axis, and 120 A for c-axis . Reflections were observed up to 25 A on x-ray photographs.

Antonie Van Leeuwenhoek, 1982, 48(3), 265 - 72
Bacillus flavothermus, a newly isolated facultative thermophile; Heinen W et al.; A sample from a hot spring on the northern island of New Zealand contained five different thermophilic bacterial strains . One strain with peculiar properties, i.e . the formation of dark yellow colonies at 30 degrees C as well as at 70 degrees C, was further characterized . It was found to be a gram-positive, facultatively aerobic, motile Bacillus species, with terminal endospores . According to the physiologic properties the strain closely resembled B . coagulans . However, two typical characteristics were contradictory to this conclusion, namely the intense yellow pigmentation of the colonies and the range of growth temperature . The latter was found to reach from 40 to 70 degrees C, with an optimum at 60 degrees C under aerobic and at 65 degrees C under anaerobic conditions . Growth at moderate temperatures was slower than at 60 degrees C, but the final cell yields were almost equal . The strain can therefore be considered as facultatively thermophilic . The pigment, which was found to be located in the cytoplasmic membrane, was spectroscopically identified as a carotenoid . Because the characteristics of this strain did not correspond with any of the Bacillus species described thus far, we concluded, that we had isolated a novel strain, for which the name Bacillus flavothermus is proposed.

J Bacteriol, 1982 Jan, 149(1), 54 - 8
Glycolipids from some extreme thermophilic bacteria belonging to the genus Thermus; Pask-Hughes RA et al.; The lipids of Thermus aquaticus YT1, Thermus thermophilus HB8, Thermus sp . strains H and J (from Icelandic hot springs), and Thermus sp . strain NH (from domestic hot water) have been investigated . Each strain contained two major components, a glycolipid and a glycophospholipid, which have been isolated and analyzed . All of the strains contained as the principal component (41 to 57% of total lipid) a diacyldiglycosyl-(N-acyl)glycosaminylglucosylglycerol, but the five glycolipids differed in carbohydrate composition . The glycophospholipid appeared to be identical in each strain and contained an N-acylglucosamine residue . The principal fatty acids were C15 and C17 branched-chain compounds . This unique polar lipid composition should be of value in the classification of other thermophiles in the genus Thermus . The exceptionally high carbohydrate content of the lipids of these extreme thermophiles may be of significance in relation to the molecular basis of thermophily.

J Bacteriol, 1982 Jan, 149(1), 237 - 46
Rapid transient growth at low pH in the cyanobacterium Synechococcus sp; Kallas T et al.; The thermophilic cyanobacterium Synechococcus sp . strain Y-7c-s grows at its maximum rate at a high pH (pH 8 and above) the does not show sustained growth below pH 6.5 . However, rapidly growing, exponential-phase cells from high-pH cultures continued to grow rapidly for several hours after transfer to pH 6.0 or 5.0 . This transient growth represented increases in mass and protein, but cells failed to complete division . Viability loss commenced well before the cessation of growth, and cells at pH 5.0 showed no net DNA synthesis . When irradiated by visible light, cells at pH 6.0 and 5.0 maintained and internal pH of 6.9 to 7.1 (determined by 31P nuclear magnetic resonance spectroscopy) and an extremely high ATP/(ATP + ADP) ratio even after growth had ceased . Cells exposed to a low pH did not show an increase in the spontaneous mutation rate, as measured by mutation to streptomycin resistance . However, cells already resistant to streptomycin were more resistant to viability loss at a low pH than the parental type . Cultures that could grow transiently at a low pH had higher rates of viability loss than nongrowing cultures in light or darkness . The retention of a high internal pH by cells exposed to a low pH suggested that a low pH acted initially on the cell membrane, possibly on solute transport.

J Bacteriol, 1982 Jan, 149(1), 229 - 36
Internal pH and ATP-ADP pools in the cyanobacterium Synechococcus sp . during exposure to growth-inhibiting low pH; Kallas T et al.; Y-7c-s Synechococcus thermophilic strain grew at its maximum rate at pH 8 and above . The growth rate of this strain was inhibited at pH 7.0 and below, and at pH 6.0 there was no sustained growth . At a suboptimal pH, high light intensity further depressed the growth rate . The inhibition of growth resulted neither from pheophytinization nor from a low chlorophyll content . At pH 5.0 a loss of viability preceded the appearance of pheophytin . Cells exposed to low, growth-inhibiting external pH levels continued to maintain a high internal pH (pH 7.1 to 7.3, as determined at moderate light intensities by 31P nuclear magnetic resonance spectroscopy) . Even during exposure to pH 4.8, cells retained a relatively high internal pH . Thus, it appeared that the inhibition of growth at low pH was not caused by acidification of the cytoplasm . Darkened cells maintained a slightly lower internal pH than irradiated cells . The ATP/(ATP + ADP) ratio decreased from 0.80 to 0.82 at pH 8.0 to about 0.6 when growth was limited by exposure to pH 6.0 or by low light intensity . It is possible, but not likely, that a limitation of the energy supply may slow or stop growth when the external pH is lowered.

Crit Rev Toxicol, 1982, 11(1), 15 - 32
Immunology of hypersensitivity pneumonitis; Stankus RP et al.; Hypersensitivity pneumonitis (extrinsic allergic alveolitis) represents a spectrum of granulomatous, interstitial, and alveolar-filling lung disorders of which farmer's lung is a classic example . A major source of offending antigens in these diseases are thermophilic actinomycetes growing in moldy vegetable matter especially Micropolyspora faeni, and members of the Thermoactinomyces genus . Acutely, hypersensitivity pneumonitis presents as cough, dyspnea and fever, with crepitant rales, leucocytosis, diffuse interstitial and alveolar pulmonary infiltrates and a restrictive-type pulmonary functional deficit . Symptoms usually begin 4 to 6 hr after exposure to large quantities of causative organic dust . Chronically, these diseases may present with the gradual onset of cough, dyspnea on exertion, fatigue, anorexia, and weight loss which may progress to pulmonary fibrosis or severe pulmonary insufficiency . While early ideas on the pathogenesis of hypersensitivity pneumonitis support the role of Type III immune complex hypersensitivity, more recent evidence attests to the important and integral role of Type IV or delayed-type hypersensitivity . It is the purpose of this review, therefore, to describe those immune mechanisms relevant to the pathogenesis of hypersensitivity pneumonitis and stress the importance of "local" pulmonary immune responsiveness.

Chromosoma, 1982, 85(1), 11 - 22
Elimination of DNA sequences during macronuclear differentiation in Tetrahymena thermophila, as detected by in situ hybridization; Yokoyama RW et al.; Previous studies have indicated that certain sequences in the micronuclear genome are absent from the somatic macronucleus of Tetrahymena (Yao and Gorovsky, 1974; Yao and Gall, 1979; Yao, submitted) . The present study used in situ hybridization to follow the elimination process during the formation of the new macronucleus . Micronuclear-specific DNA cloned in recombinant plasmids was labelled with 3H and hybridized to cytological preparations of T . thermophila at various stages of conjugation . Despite a smaller size and lower DNA content, the micronucleus has more hybridization than the mature macronucleus . Hybridization initially increased in the anlage (newly developing macronucleus) to reach a maximal level right after the old macronuclei has disappeared . The hybridization in the anlage than decreased to a significant extent prior to the first cell division . The results suggest that the micronuclear-specific sequence is first replicated a few rounds before it is eliminated from the anlage, and the elimination process occurs without nuclear division.

Tokai J Exp Clin Med, 1982, 7 Suppl, 15 - 22
Image analysis of electron micrographs of ATPase (Coupling Factor TF1) from thermophilic bacteria and luminal epithelium of mouse urinary bladder; Wakabayashi T et al.; Electron micrographs of two dimensional array of H+-ATPase (Coupling Factor TF1 from the thermophilic bacteria) and luminal epithelial protein of urinary bladder were obtained using negative staining method and freeze-fracture method, respectively . Images which showed good optical diffraction pattern were digitized by a computer-linked flat-bed two dimensional microdensitometer and processed digitally . The results of translational computer noise filtering showed that the outline of TF1 molecules looks like a hexagon or an asterisk in the presence of sodium azide which is a specific inhibitor of TF1 or AMPPNP which is an unsplitable analogue of ATP, respectively . The luminal epithelial protein also looks like a hexagon . Rotational harmonic analysis was carried out to examine the rotational symmetry of the filtered images of TF1 . It was found that 2-fold or 6-fold symmetry is dominant in the presence of sodium azide or AMPPNP, respectively.

Nucleic Acids Symp Ser, 1982, (11), 135 - 8
A mode of action of RNase CI toward the various RNAs; Uchida T et al.; A mode of action of RNase CI toward the various native RNAs was examined . The mixture of tRNA from Thermus thermophilus HB8 were exhaustively cleaved by RNase CI to produce 90% of acid-soluble fraction . Among the minor nucleotides s2T were recognized by this enzyme . RNase CI generates a linear viroid molecule by introducing one single nick into viroid circular molecule . Double stranded RNA from RNA virus, such as Cytoplasmic polyhedrosis virus (CPV) from silk worm, was cleaved at some specific sites to produce more than ten of the discrete bands electrophoretically.

Nucleic Acids Res, 1981 Dec 21, 9(24), 6795 - 804
Purification and characterization of two new modification methylases: MClaI from Caryophanon latum L and MTaqI from Thermus aquaticus YTI; McClelland M; A method for detecting Type II modification methylases and determining their methylation site by assaying the ability of methylated DNA to be cleaved by heterologous restriction enzymes is described and applied to the isolation of the restriction modification methylases from Thermus thermophilus HB8, Thermus aquaticus YTI and Caryophanon latum L . M.TaqI is shown to have a methylation specificity identical to M.ThI (TCGmeA) . M.ClaI methylates at adenine and protects a subset of TthI sites indicating that it methylates the sequence ATCGmeAT . Methylation by M.ThI also protects against cleavage by SalI, XhoI and at some HindII, AccI and MboI sites.

Wien Klin Wochenschr, 1981 Dec 11, 93(23), 729 - 33
{Some observations on farmer's lung (author's transl)}; Ebner H et al.; Sera from 72 patients--29 females, 43 males--with symptoms suggestive of farmer's lung were tested by the Ouchterlony double diffusion technique using 10 different antigen solutions (extracts from mycetes and thermophilic actinomycetes, pigeon serum, extracts from pigeon droppings and Sitophilus granarius) . In 23 cases precipitating antibodies were observed and Micropolyspora faeni was the most common antigen responsible for positive reactions . In 17 seropositive patients the diagnosis of exogen allergic alveolitis was established by clinical and laboratory findings . Measurements of immunoglobulins in these sera showed pronounced elevation of IgG and slightly elevated levels of IgA; IgM, IgE and C 3 c levels were in the normal range . The results are discussed with regard to the classification of farmer's lung as an occupational disease in Austria since December 30th, 1980.

Eur J Biochem, 1981 Dec, 121(1), 155 - 62
Properties of native and nicked elongation factor Tu from Thermus thermophilus HB 8; Gulewicz K et al.; Two alternative procedures for the isolation of the elongation factor Tu from Thermus thermophilus were compared and the properties of a specifically nicked EF-Tu . GDP were examined in detail . Although the native elongation factor possessed similar catalytic activities in all reactions investigated as the protein isolated by Arai et al . {Eur . J . Biochem . 92, 509-519 and 521-531 (1978)} it could not be crystallized . The nicked EF-Tu, consisting of two associated fragments with molecular weights of 41000 and 8000 respectively, was active in binding GDP, GTP and in the formation of Phe-tRNAPhe . EF-Tu . GTP ternary complex . However, it did not promote poly(U)-dependent synthesis of polyphenylalanine on Escherichia coli ribosomes . The isolated fragment of a molecular weight of about 41000 did not bind GDP . This activity could be reconstituted with the supplement of the small 8000-Mr fragment . It is demonstrated that, in contrast to the native EF-Tu, the nicked EF-Tu forms high-molecular-weight aggregates . Cleavage of the polypeptide chain of EF-Tu from T . thermophilus stimulates the crystallization of this protein.

Proc Natl Acad Sci U S A, 1981 Dec, 78(12), 7614 - 8
Phenylketonuric Tetrahymena: phenylalanine hydroxylase mutants and other tyrosine auxotrophs; Sanford YM et al.; Nineteen tyrosine auxotrophs of the ciliated protozoan Tetrahymena thermophila have been isolated and biochemically examined . These mutants are defective in the conversion of phenylalanine to tyrosine; this is analogous to the defect that causes phenylketonuria in humans . After nitrosoguanidine mutagenesis and self-fertilization, progeny clones were screened for tyrosine auxotrophy and positively identified by using growth tests and in vivo radiometric assays for phenylalanine-to-tyrosine conversion . Mutants in one complementation group (locus) lacked phenylalanine hydroxylase activity; mutants in three other loci appeared to be deficient in the unconjugated pteridine cofactor that is necessary for the function of the hydroxylase . Another mutant lacked the dihydropteridine reductase activity required to regenerate the reduced form of the pteridine cofactor . Because hydroxylation of tyrosine to dopa and of tryptophan to 5-hydroxytryptophan may require the same cofactor and pterin reductase as phenylalanine hydroxylase, these mutants may also prove useful for the study of the role of catecholamines and serotonin, substances known to be present in Tetrahymena.

Appl Environ Microbiol, 1981 Dec, 42(6), 1023 - 8
Recovery of indigenous enteroviruses from raw and digested sewage sludges; Goddard MR et al.; We examined different types of raw sewage sludge treatment, including consolidation, anaerobic mesophilic digestion with subsequent consolidation, and aerobic-thermophilic digestion . Of these, the most efficient reduction in infectious virus titer was achieved by mesophilic digestion with subsequent consolidation, although a pilot-scale aerobic-thermophilic digester was extremely time effective, producing sludges with similarly low virus titers in a small fraction of the time . Although none of the treatments examined consistently produced a sludge with undetectable virus levels, mesophilic digestion alone was found to be particularly unreliable in reducing the levels of infectious virus present in the raw sludge.

Nucleic Acids Res, 1981 Nov 11, 9(21), 5825 - 43
Nuclease sensitivity of chromatin containing active genes: kinetic analyses utilizing continuous elution of digestion products from an ultrafiltration cell; Vavra KJ et al.; Methods have been developed to analyze the kinetics of digestion of chromatin by nucleases . Radioactively labeled nuclei were incubated with enzyme in an ultrafiltration apparatus and digestion rates of different chromatin samples were computed employing a least-squares curve fitting technique to fit the data to zero-order and/or first-order kinetic models . These methods allow detailed kinetic analyses on small amounts of chromatin . Two biological systems were studied . 1) Tetrahymena thermophila macronuclei and micronuclei were compared; these nuclei differ in their transcriptional activities . 2) Ribosomal DNA (rDNA) of Tetrahymena pyriformis, approximately 60% of which codes for rRNA, can be preferentially labeled during starvation-refeeding; its digestion kinetics relative to bulk chromatin were studied . DNase I digested 20-40% of the macromolecular DNA about 3 times faster than bulk macronuclear or micronuclear DNA, and 60-80% of ribosomal gene-containing chromatin about 5 times faster than bulk chromatin . Filter hybridization studies of the DNAase I sensitivity of tRNA, 5S RNA, and ribosomal genes yielded similar results . These data are consistent with the observation that transcribed genes are especially sensitive to attach by DNase I and suggest that activated chromatin structure as probed by extensive DNase I digestion is the same in higher and lower eucaryotes for genes transcribed by all three RNA polymerases . Digestion kinetics of micrococcal nuclease were found to depend on the digestion conditions employed . These two biological systems and the methods we have developed should facilitate analyses of the factors responsible for maintaining an active chromatin structure.

Int J Pept Protein Res, 1981 Nov, 18(5), 430 - 42
Protein thermostability . Correlations between calculated macroscopic parameters and growth temperature for closely related thermophilic and mesophilic bacilli; Merkler DJ et al.; The amino acid composition of more than 20 enzymes and protein from various closely related mesophilic and thermophilic micro-organisms (esp . Bacillus) have been used to calculate a variety of macroscopic parameters . These included the hydrophobic index (H phi ), the ratio of polar to non-polar volumes (rho), the ratios of Arg/(Arg + Lys), and (Arg + Lys) or (Glx + Asx) to total amino acids, % H-bonding amino acids, % alpha-helix- or beta-sheet-forming amino acids, the theoretical melting temperature (TCalcm), the total volume to total amino acid ratio (VR), and the % non-polar residues (NPS) . In contrast to previous similar comparisons with proteins from divergent sources, it was found that thermophilic vs mesophilic proteins from the same genus show correlations between thermostability and increased H phi, decreased rho, and increased Arg/(Arg + Lys), as well as increased alpha-index and beta-index . Weaker correlations were seen for VR, TCalcm, aliphatic index, and NPS, all derived from, or related to, H phi . No correlations existed for the other calculated parameters . These results are consistent with recent results of Argos et al . (1979) {Biochemistry 18, 5698-5703} on sequence analyses of glyceraldehyde-3-P dehydrogenases, where thermophilic proteins showed multiple amino acid replacements that caused increased internal hydrophobicity and increased external polarity . No trends were observed in any of the parameters calculated from amino acid compositions for crude cytoplasmic protein extracts from mesophilic vs thermophilic Bacilli.

J Biochem (Tokyo), 1981 Nov, 90(5), 1521 - 7
Molecular multiplicity of nuclease TT1 from Thermus thermophilus HB8; Takahashi M et al.; Purified nuclease TT1 from Thermus thermophilus HB8 has multimolecular weight forms, each of which is composed of three different subunits, alpha (10.8 x 10(4)), beta (7.8 x 10(4)), and gamma (4.1 x 10(4)) . The molecular weights of this enzyme were estimated by gel filtration, polyacrylamide gel electrophoresis and equilibrium sedimentation . It was found that most of the enzyme has a molecular weight of about 22 x 10(4) being a monomer having the subunit composition of alpha beta gamma . The remaining part of the enzyme has larger molecular weights and is considered to be size-isomers of alpha beta gamma . The alpha-helical content, 5.5--6.5%, and the beta-structure, about 28%, were estimated from the CD spectrum at 4 degrees C.

Nucleic Acids Res, 1981 Oct 10, 9(19), 4909 - 17
Sequence and secondary structure of the colicin fragment of Bacillus stearothermophilus 16S ribosomal RNA; Van Charldorp R et al.; The sequence and the position of post-transcriptionally modified residues of the 3' -terminal end of Bacillus stearothermophilus 16S ribosomal RNA have been determined from the fragment that is cleaved off by bacteriocin treatment . The fragment contains 52 nucleotides, as compared to the 49 nucleotides of the corresponding fragment from E . coli ribosomes, The additional nucleotides are present in the sequence UCU very next to the 3' -terminus as was published earlier (1) . The remainder of the sequence is identical to the one of E . coli except at six positions, due to the UV melting properties of the colicin fragment from B . stearothermophilus in comparison to the same fragment of E . coli show that the RNA from the thermophile has a more stable secondary structure.

Cell, 1981 Oct, 26(1 Pt 1), 47 - 56
Regulation of ribosome phosphorylation and antibiotic sensitivity in Tetrahymena thermophila: A correlation; Hallberg RL et al.; Tetrahymena thermophila cells transferred from growth medium into a dilute salt (starvation) medium shortly (approximately 6-8 hrs) become more resistant to the in vivo inhibitory effects of the antibiotics cycloheximide, tetracycline and emetine . They also be come more sensitive to the inhibitory effects of paromomycin and anisomycin . By comparing ribosomes from growing and starved cells we have found that for at least two of these drugs differences between growing cell and starved cell ribosomes exist with respect to drug-ribosome interactions . In addition, we found that isolated monosomic ribosomes from starved cells are more resistant to thermal denaturation than are monosomic ribosomes from growing cells . The kinetics of all these changes following transfer of growing cells to starvation medium is the same and correlates with a change in the extent of phosphorylation of a single small subunit ribosomal protein . As judged by our in vitro assays, enzymatic removal of this phosphate converts "starved cell" ribosomes into "growing cell" ribosomes . We have extended these studies to show that the phenomenon of drug adaptation in Tetrahymena, at least with respect to cycloheximide, is associated with this ribosome phosphorylation.

J Cell Sci, 1981 Oct, 51, 241 - 53
Cytoskeleton-related structures in tetrahymena thermophila: microfilaments at the apical and division-furrow rings; Jerka-Dziadosz M; A ring consisting of microfilaments was found in the apical region of Tetrahymena thermophila wild-type strain B and janus mutant . This ring, about 0.4 micrometer wide and 0.2 micrometer thick, is located at the bases of the anterior, non-ciliated basal bodies of the apical ciliary couplets . The apical ring is made of fine filaments showing a banded pattern, the distance between bands depending on the fixation procedure and ranging from 30-200 nm . The bands are made of small beads fastened to the filaments . The microfilaments of the apical ring are attached to the bases of the basel bodies . No connection with the cell membrane was found . In dividing cells in the incipient furrow region of filamentous band originates from the epiplasmic fibrogranular meshwork . This contractile ring is about 0.4 micrometer wide and 0.8 micrometer thick . It is formed by circumferentially aligned microfibrils . During constriction the contractile ring remains associated with the epiplasmic layer, which in turn adheres to the inner alveolar membrane . The microfilaments of both the apical and the division-furrow rings have diameters ranging from about 3.8-7.I nm.

Nucleic Acids Res, 1981 Sep 25, 9(18), 4557 - 62
Nucleotide sequence of cytoplasmic initiator tRNA from Tetrahymena thermophila; Kuchino Y et al.; The total primary structure of cytoplasmic initiator tRNA from Tetrahymena thermophila mating type IV, was determined by post labeling techniques . The sequence is pa-G-C-A-G-G-G-U-m1G-G-C-G-A-A-A-D-Gm-G-A-A-U-C-G-C-G-U-Psi-G-G-G-C-U-C-A-U-t6A -A-C-Psi-C-A-A-A-A-m7G-U-m5C-A-G-A-G-G-A-Psi-C-G-m1A-A-A-C-C-U-C-U-C-U-C-U-G-C- U-A-C-C-AOH . The nucleotide residue in the position next to the 5'-end of the anticodon of this tRNA (residue No . 33) is uridine instead of cytidine, which has been found in cytoplasmic initiator tRNAs from multicellular eukaryotic organisms . The sequence of three consecutive G-C base pairs in the anticodon stem common to all other cytoplasmic initiator tRNAs is disrupted in this tRNA; namely, the cytidine at residue 40 in this region is replaced by pseudouridine in Tetrahymena initiator tRNA.

Biochim Biophys Acta, 1981 Sep 15, 661(1), 158 - 63
Phosphofructokinases from Lactobacteriaceae . II . Purification and properties of phosphofructokinase from Streptococcus thermophilus; Simon WA et al.; Phosphofructokinase (ATP : D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) from Streptococcus thermophilus has been purified . It is a tetramer composed of identical subunits of molecular weight 36 000 and exhibits Michaelis-Menten kinetics . Compared to the phosphofructokinases from taxonomically related bacteria, the enzyme from S . thermophilus is more stable at high temperatures . In addition, it has been demonstrated that the phosphofructokinases from lactobacteria and also from Bacillus stearothermophilus show immunologic cross-reaction . In spite of the significantly different kinetic properties and the different thermostability of these enzymes, this finding indicates great structural resemblance.

Mol Cell Biol, 1981 Sep, 1(9), 865 - 70
Control of initiation and elongation of cilia during ciliary regeneration in Tetrahymena; Hadley GA et al.; Tetrahymena thermophila strain B could regenerate approximately 10% of its somatic ciliary mass in concentrations of cycloheximide believed to block all cytoplasmic protein synthesis . A quantitative study of the relative numbers and lengths of cilia regenerated in the presence and absence of cycloheximide under a variety of conditions suggested that specific initiation and elongation protein factors are involved in the control of ciliary morphogenesis in Tetrahymena.

Biokhimiia, 1981 Sep, 46(9), 1576 - 84
{Isolation and properties of DNA-polymerase from the extreme thermophilic bacterium Thermus flavus}; Kaledin AS et al.; A DNA-polymerase from the extreme thermophilic bacteria Thermus flavus has been isolated . The five-step purification procedure resulted in an electrophoretically homogeneous enzyme with molecular weight of 66000 . The isolated enzyme is thermostable and a temperature optimum on the DNA templates at 70 degrees and that on RNA templates at 50 degrees . The enzyme does not contain contaminant endo- and exonuclease activities . The maximal activity of the enzyme requires the presence of template, four deoxyribonucleoside triphosphates and monovalent and bivalent cations in the incubation mixture . The enzyme is highly active when "activated" DNA, poly(dA)-poly(dT), poly(dA)-oligo(dT) 10 and poly(rA)-oligo(dT)10 are used as templates, moderately active on single-stranded and double-stranded DNAs and inactive on poly(rC)-oligo(dG)12-18 and native RNA molecules.

Sabouraudia, 1981 Sep, 19(3), 179 - 85
Nutritional physiology of pathogenic species of thermophilic mucor; Ogundero VW; Some aspects of the nutritional physiology of zoopathogenic Mucor pusillus Lindt and Mucor miehei Cooney & Emerson were studied at 37 degrees C . Only asexual spores (sporangiospores) were produced on all the media used . A pH range of 5.0-6.0 was found to be best for the growth and sporulation of both species . The fungi were able to utilize various sources of carbon and nitrogen tested except L-sorbose and DL-tryptophan on which no growth was recorded . Of the carbon sources, the best results were obtained with dextrin and starch while of the nitrogen sources, casein hydrolysate was most readily utilized . Both species gave measurable growth with sporulation on soluble carboxymethyl cellulose . A glucose and DL-glutamine concentration range 15.0-20.0 gl-1 and 5.0-7.5 gl-1 respectively was optimal for growth and sporulation of these fungi . A C/N ratio of 15.0: 5.0 gl-1 was optimal for sporulation and 20.07: 7.5 gl-1 for mycelial growth of both species.

Biochim Biophys Acta, 1981 Aug 28, 670(1), 39 - 46
Superoxide dismutase from the Archaebacterium Thermoplasma acidophilum; Searcy KB et al.; Thermoplasma acidophilum is a mycoplasma-like thermophilic organism that has been classified with the archaebacteria . It has a single superoxide dismutase (superoxide : superoxide oxidoreductase, EC 1.15.1.1) which is composed of four identically sized subunits . It has a metal content per molecule of two atoms of iron and probably one of zinc and a molecular weight of 82 000 . The amino acid composition is rich in tryptophan and is typical of the manganese or iron superoxide dismutases found in other prokaryotes . However, the enzyme is resistant to denaturation by chloroform plus ethanol, by sodium dodecyl sulfate plus urea or by heat . In these respects it resembles the copper-zinc superoxide dismutase of eukaryotes . It is suggested that the enzyme may belong to a new group of superoxide dismutases.

Genetics, 1981 Aug, 98(4), 747 - 62
Further evidence for lack of gene expression in the Tetrahymena micronucleus; Mayo KA et al.; Certain galA mutations in the ciliated protozoan Tetrahymena thermophila confer an almost total loss of galactokinase activity in homozygotes . Heterokaryons have been constructed that are homogeneous for the galA1 mutation in the (45n) macronucleus, but which contain a galA+ (2n) micronucleus . Soluble cell extracts prepared from these heterokaryons have been assayed for galactokinase activity, using a radiometric assay for the conversion of galactose to galactose-1-phosphate (gal-1-P) . No galactokinase activity attributable to the micronuclear genes is observed in such heterokaryons . These results, obtained with the galA1 marker, provide the first direct, quantitative evidence for the lack of micronuclear (germ line) gene expression in Tetrahymena during vegetative growth, and substantiate the predictions of previous phenotypic observations on heterokaryons and autoradiographic studies of micronuclear RNA synthesis . The generality of this conclusion will be established in the future when other enzymically assayable mutations become available for similar studies.

J Cell Sci, 1981 Aug, 50, 407 - 18
External factors limiting the multiplication potential of Tetrahymena; Hofmann E et al.; By variation of nutritional and other external conditions we have determined the factors that limit the multiplication rate and the culture growth in Tetrahymena thermophila . The enriched synthetic medium of Kidder & Dewey (1951), a culture temperature of 29 degrees C, and aeration by agitation were chosen as reference conditions . The final cell density is increased by and proportional to the amount of the complete set of nutrients . Testing single nutritional factors or groups of them revealed that only nitrogen sources yield higher cell densities . But none of them or any combination is as capable of increasing the cell density as the complete medium . Therefore, the medium has to be considered as well balanced . Ammonia, cell density, O2 supply, and pH have been excluded as factors limiting the capacity for multiplication . There are no known factors promoting or inhibiting culture growth.

Eur J Biochem, 1981 Aug, 118(2), 423 - 7
Influence of growth temperature on structure, thermostability and kinetic properties of the ATPase coupling factor AF1 of a thermophilic blue-green alga (cyanobacterium); Wolf M et al.; The coupling factor AF1 isolated from cells of the thermophilic blue-green alga Mastigocladus laminosus grown at 40 degrees C, 50 degrees C and 60 degrees C is investigated and compared with the chloroplast coupling factor CF1 . It is demonstrated that the structure of AF1 is affected by the growth temperature . The AF1 from 60 degrees C shows a split alpha-band in dodecylsulfate/polyacrylamide gel electrophoresis which is not observed in AF1 from 40 degrees C or from 50 degrees C . Only the AF1 from 60 degrees C aggregates with allophycocyanin . The AF1 from 60 degrees C is stable up to 85 degrees C for 60 min whereas the AF1 from 40 degrees C and 50 degrees C denature between 65 degrees C and 70 degrees C . CF1 is much less stable . In contrast to the structure, the kinetic properties of the coupling factor are not influenced by the growth temperature . Furthermore, the reaction rates of all three AF1 preparations have the same temperature optimum which jis also identical to that of CF1 . The kinetic properties of the AF1 from 50 degrees C were determined in more detail . In the absence of ADP the saturation curve for ATP shows Michaelis-Menten kinetics with Km = 480 micromol and V = 30 micromol ATP hydrolyzed X mg protein-1 X min-1 . With the addition of ADP this curve becomes sigmoidal and V decreases . This behaviour and the Hill values suggest an allosteric enzyme with two active sites acting cooperatively . ADP as a product of the ATPase reaction inhibits the enzyme . The inhibition is not simply competitive, it also influences the cooperativity of the active sites.

Nucleic Acids Res, 1981 Jul 24, 9(14), 3531 - 43
Replication of the extrachromosomal ribosomal RNA genes of Tetrahymena thermophilia; Cech TR et al.; Cultures of Tetrahymena thermophila were deprived of nutrients and later refed with enriched medium to obtain partial synchrony of DNA replication . Preferential replication of the extrachromosomal, macronuclear ribosomal RNA genes (rDNA) was found to occur at 40-80 min after refeeding . The rDNA accounted for one half of the label incorporated into cellular DNA during this period . Electron microscopy of the purified rDNA showed 1% replicative intermediates . Their structure was that expected for bidirectional replication of the linear rDNA from an origin or origins located in the central nontranscribed region of the palindromic molecule . Similar forms had previously been observed for the rDNA of a related species, Tetrahymena pyriformis . The electron microscopic data was consistent with an origin of replication located approximatley 600 base pairs from the center of the rDNA of T . thermophila, in contrast to a more central location in the rDNA of T . pyriformis . One implication of an off-center origin of replication is that there are two such sequences per palindromic molecule.

Nucleic Acids Res, 1981 Jul 24, 9(14), 3523 - 9
Is wheat mitochondrial 5S ribosomal RNA prokaryotic in nature?
Gray MW, Spencer DF.
Kuntzel et al . (1981) (Nucleic Acids Res . 9, 1451-1461) recently concluded that the sequence of wheat mitochondrial 5S rRNA is significantly more related to prokaryotic than to eukaryotic 5S rRNA sequences, and displays an especially high affinity to that of the thermophilic Gram-negative bacterium, Thermus aquaticus . However, the sequence on which this conclusion was based, although attributed to us, differs in several places from the one determined by us . We show here that the correct sequence (Spencer, D.F., Bonen, L . and Gray, M.W . (1981) Biochemistry, in press) does not support the conclusions of Kuntzel et al . about potential secondary structure in wheat mitochondrial 5S rRNA and its phylogenetic significance . We further show that when the wheat mitochondrial 5S rRNA sequence is matched against published alignments for E . coli, T . aquaticus, and wheat cytosol 5S rRNAs, the mitochondrial sequence shows no greater homology to the T . aquaticus sequence than to the E . coli sequence, and only slightly more homology to these two sequences than to wheat cytosol 5S rRNA . This analysis confirms our original view (Biochemistry, in press) that wheat mitochondrial 5S rRNA is neither obviously prokaryotic nor eukaryotic in nature, but shows characteristics of both classes of 5S rRNA, as well as some unique features.

Mol Cell Biol, 1981 Jul, 1(7), 600 - 8
Deoxyribonucleic acid methylation and chromatin organization in Tetrahymena thermophila; Pratt K et al.; Deoxyribonucleic acid (DNA) of the transcriptionally active macronucleus of Tetrahymena thermophila is methylated at the N6 position of adenine to produce methyladenine (MeAde); approximately 1 in every 125 adenine residues (0.8 mol%) is methylated . Transcriptionally inert micronuclear DNA is not methylated (< or = 0.01 mol% MeAde; M . A . Gorovsky, S . Hattman, and G . L . Pleger, J . Cell Biol . 56:697-701, 1973) . There is no detectable cytosine methylation in macronuclei in Tetrahymena DNA (< or = 0.01 mol% 5-methylcytosine) . MeAde-containing DNA sequences in macronuclei are preferentially digested by both staphylococcal nuclease and pancreatic deoxyribonuclease I . In contrast, there is no preferential release of MeAde during digestion of purified DNA . These results indicate that MeAde residues are predominantly located in "linker DNA" and perhaps have a function in transcription . Pulse-chase studies showed that labeled MeAde remains preferentially in linker DNA during subsequent rounds of DNA replication; i.e., there is little, if any, movement of nucleosomes during chromatin replication . This implies that nucleosomes may be phased with respect to DNA sequence.

Can J Microbiol, 1981 Jul, 27(7), 720 - 8
The transmembrane electrical potential and intracellular pH in methanogenic bacteria; Jarrell KF et al.; The magnitudes of the electrical potential and proton gradient in Methanospirillum hungatei GP1 and Methanobacterium thermoautotrophicum were determined . No delta pH (inside alkaline) could be demonstrated in either organism suspended in growth media at normal growth pH values by the distribution of 5,5-dimethyl-2,4-oxazolidinedione (DMO), butyrate, propionate, or methylamine . The internal pH, estimated to be approximately 6.7 under our growth conditions, was not constant, but varied as the external pH was adjusted . However, the internal pH was always more neutral than the external pH (except at pH 6.7 where the two were equal) . The distribution of triphenylmethylphosphonium cation, in the presence of tetraphenylboron anion, gave estimates of 119 and 79 mV (interior negative) for the electrical potentials of the thermophile and mesophile, respectively, for cells suspended in a phosphate buffer (pH 7.0) . The uptake of 86Rb+, in the presence of valinomycin, gave similar results for M . thermoautotrophicum, ranging from 143 mV (at pH 5.8) to 120 mV (at pH 8.0) . Electrical potentials compared to the size of the respective K+ gradients, maintained between the cytoplasm and growth medium . The results are interpreted in terms of proton efflux and monovalent cation antiport activities at the cytoplasmic membrane, with possible proton pumping at the site of internal vesicles.

Biochimie, 1981 Jul, 63(7), 629 - 39
Purification and properties of an endo-beta-1,4-glucanase from Clostridium thermocellum; Petre J et al.; The extracellular cellulolytic enzymes of the thermophilic anaerobe Clostridium thermocellum occur as a protein complex or aggregate which, until now, has not been resolved into individual enzyme components . By using QAE-Sephadex A50 chromatography in the presence of 6 M urea, it was possible to split the complex into distinct protein fractions . One of these fractions contained an endo-beta-1,4-glucanase which was isolated at a high degree of purity and was identified by its ability to hydrolyze trinitrophenylated carboxymethylcellulose . The enzyme is of monomeric nature, with a molecular weight of 56,000 . It has an isoelectric pH of 6.2 and an optimum pH of 6.0 . It hydrolyzed carboxymethylcellulose and, at a slower rate, cellulose powder . The major end products of cellulose degradation are glucose, cellobiose and cellotriose; cellotetrose is formed as an intermediate product . No specific small molecular weight activator or inhibitor was found except cellobiose and, to a lesser extent, glucose, which at high concentrations partially inhibit the activity of the enzyme . The temperature dependence of the enzyme is related to the thermophilic character of the producing microorganism.

Biochemistry, 1981 Jun 23, 20(13), 3828 - 33
Histone phosphorylation in macro- and micronuclei of Tetrahymena thermophila; Allis CD et al.; The patterns of histone phosphorylation in amitotically dividing, transcriptionally active macronuclei and in mitotically dividing, transcriptionally inert micronuclei of the ciliated protozoan Tetrahymena thermophila have been analyzed . Taken together, the major phosphorylation events in these two nuclei and their dependence on cell growth and/or division are remarkably similar to those in mammalian cells . Phosphorylation of H1-type proteins occurs in both nuclei and is positively correlated with growth and/or division . Phosphorylation of histone H3 is also positively correlated with growth and/or division but occurs only in micronuclei . Phosphorylation of histone H2A is relatively independent of growth state and occurs largely, if not exclusively, in macronuclei . Given the unique partition of nuclear functions between macro- and micronuclei, these results, coupled with previously reported temporal correlations between specific histone phosphorylations and cell cycle events in mammalian cells {Gurley, L . R., Tobey, R . A., Walters, R . A., Hildebrand, C . E., Hohmann, P . G., D'Anna, J . A., Barham, S . S., & Deaven, L . L . (1978a) in Cell Cycle Regulation (Jeter, J . R., Cameron, J . L., Padilla, G . M . & Zimmerman, A . M . Eds.) pp 37-60, Academic Press, New York}, allow insights into the functions of histone phosphorylations . Specifically, a nonmitotic function for extensive H1 phosphorylation and a unique mitotic function for H3 phosphorylation are clearly indicated . A new role for H2A phosphorylation in the regulation of transcriptional activity is also proposed.

J Biol Chem, 1981 Jun 10, 256(11), 5857 - 9
Characterization of crystals of tetrameric manganese superoxide dismutase from Thermus thermophilus HB8; Stallings WC et al.; The tetrameric manganese superoxide dismutase from the extreme thermophile Thermus thermophilus HB8 crystallizes in space group P41212 (or its enantiomorph) with a = b = 147.5 A, c = 55.9 A . The diffraction patterns extent to 1.4 A, and the crystals are very resistant to decay induced by x-irradiation . Measurements of the crystal density in Ficoll gradients are consistent with an asymmetric unit containing the entire tetramer (Mr = 80,000).

J Biol Chem, 1981 Jun 10, 256(11), 5675 - 82
Studies on the secondary structure of 5.8 S rRNA from a thermophile, Thermomyces lanuginosus; Wildeman AG et al.; The nucleotide sequence of ribosomal 5.8 S RNA from a thermophilic fungus, Thermomyces lanuginosus, was determined to be (formula: see text) . The secondary structure was probed under a variety of ionic conditions using limited pancreatic and T1 ribonuclease digestion and rapid gel sequencing techniques . The results generally supported the "burp gun" model previously proposed for all 5.8 S rRNAs (Nazar, R . N., Sitz, T . O., and Busch, H . (1975) J . Biol . Chem . 250, 8591-8597) and were inconsistent with a recently suggested "cloverleaf" configuration (Luoma, G . A., and Marshall, A . G . (1978) Proc . Natl . Acad . Sci . U.S.A . 75, 4901-4905) . Theoretical considerations of the overall stability also appear to favor the former estimate . As previously observed with other 5.8 S RNAs the sequence of T . lanuginosus RNA is strikingly homologous with other species; it differs in only 13 positions from that of yeast . When compared to yeast, the differences appear not to contribute to the stability of the secondary structure but probably lead to a more stable 5.8 S-25 S rRNA interaction in the large ribosomal subunit . A general comparison of T . lanuginosus 5.8 S RNA with all known 5.8 S RNA sequences indicates that, although modified nucleotides differ significantly between species, they are always located in four highly conserved regions of the 5.8 S molecule, probably contributing to the unique character of these very essential sequences.

J Biochem (Tokyo), 1981 Jun, 89(6), 1903 - 12
The quaternary structure of DNA-dependent RNA polymerase; Tsuji S et al.; The crystals of RNA polymerase of T . thermophilus were examined by electron microscopic observation of the negatively stained and sectioned materials . Three types of crystals were observed: ordered aggregates (Type I), cylindrical duplei (Type II), and plane (Type III) forms . It was deduced mainly from sectioned images that Type I crystal is a precursor or a premature form of Type II crystal . The III corresponds to the flattened layer of type II . In Type II crystal the enzyme molecules are arranged in an orderly two-dimensional lattice and thus we could analyze the molecular structure by optical filtering of the negatively stained images . On the basis of these results, a quaternary structure is proposed for RNA polymerase.

Hoppe Seylers Z Physiol Chem, 1981 Jun, 362(6), 611 - 28
The complete amino acid sequence of both subunits of allophycocyanin, a light harvesting protein-pigment complex from the cyanobacterium Mastigocladus laminosus; Sidler W et al.; The amino acid sequences of the alpha- and beta-subunit of allophycocyanin, a water-soluble light-harvesting protein-pigment complex from the thermophilic cyanobacterium Mastigocladus laminosus have been determined . The alpha-chain consists of 160 amino acid residues and the beta-chain of 161 amino acid residues . The homology of the alpha- and beta-chains is 37% . A comparison with C-phycocyanin reveals that the second chromophore of the C-phycocyanin beta-subunit is attached to an inserted peptide of 10 amino acid residues at position 151-160.

Mol Cell Biol, 1981 Jun, 1(6), 535 - 43
Two separate regions of the extrachromosomal ribosomal deoxyribonucleic acid of Tetrahymena thermophila enable autonomous replication of plasmids in Saccharomyces cerevisiae; Kiss GB et al.; Plasmids containing the nontranscribed central and terminal, but not the coding, regions of the extrachromosomal ribosomal deoxyribonucleic acid (rDNA) of Tetrahymena thermophila are capable of autonomous replication in Saccharomyces cerevisiae . These plasmids transform S . cerevisiae at high frequency; transformants are unstable in the absence of selection, and plasmids identical to those used for transformation were isolated from the transformed yeast cells . One plasmid contains a 1.85-kilobase Tetrahymena DNA fragment which includes the origin of bidirectional replication of the extrachromosomal rDNA . The other region of Tetrahymena rDNA allowing autonomous replication of plasmids in S . cerevisiae is a 650-base pair, adenine plus thymine-rich segment from the rDNA terminus . Neither of these Tetrahymena fragments shares obvious sequence homology with the origin of replication of the S . cerevisiae 2-microns circle plasmid or with ars1, an S . cerevisiae chromosomal replicator.

Biophys J, 1981 Jun, 34(3), 465 - 98
Structural features and the reaction mechanism of cytochrome oxidase: iron and copper X-ray absorption fine structure; Powers L et al.; X-ray edge absorption of copper and extended fine structure studies of both copper and iron centers have been made of cytochrome oxidase from beef heart, Paracoccus dentrificans, and HB-8 thermophilic bacteria (1-2.5 mM in heme) . The desired redox state (fully oxidized, reduced CO, mixed valence formate and CO) in the x-ray beam was controlled by low temperature (-140 degrees C) and was continuously monitored by simultaneous optical spectroscopy and by electron paramagnetic resonance (EPR) monitoring every 30 min of x-ray exposure . The structure of the active site, a cytochrome a3-copper pair in fully oxidized and in mixed valence formate states where they are spin coupled, contains a sulphur bridge with three ligands 2.60 +/- 0.03 A from Fea3 and 2.18 +/- 0.03 A from Cua3 . The distance between Fea3 and Cua3 is 3.75 +/- 0.05 A, making the sulphur bond angle 103 degrees reasonable for sp3 sulphur bonding . The Fea3 first shell has four typical heme nitrogens (2.01 +/- 0.03 A) with a proximal nitrogen at 2.14 +/- 0.03 A . The sixth ligand is the bridging sulphur . The Cua3 first shell is identical to oxidized stellacyanin containing two nitrogens and a bridging sulphur . Upon reduction with CO, the active site is identical to reduced stellacyanin for the Cua3 first shell and contains the sulphur that forms the bridge in fully oxidized and mixed valence formate states . The Fea3 first shell is identical to oxyhemoglobin but has CO instead of O2 . The other redox centers, Fea and the other "EPR detectable" Cu are not observed in higher shells of Fea3 . Fea has six equidistant nitrogens and Cua has one (or two) nitrogens and three (or two) sulphurs with typical distances; these ligands change only slight on reduction . These structures afford the basis for an oxygen reduction mechanism involving oxy- and peroxy intermediates.

J Bacteriol, 1981 Jun, 146(3), 1091 - 7
Isolation and characterization of antibiotic resistance plasmids from thermophilic bacilli and construction of deletion plasmids; Imanaka T et al.; Ten plasmids were isolated as covalently closed circular deoxyribonucleic acid from antibiotic-resistant thermophilic bacteria . Of the 10 plasmids tested, 2 could transform Bacillus subtilis, yielding resistance to specific antibiotics . Plasmid pTB20 (2.8 X 10(6) daltons, approximately 24 copies per chromosome) specifies resistance to tetracycline (Tcr), whereas pTB19 (17.2 X 10(6) daltons, approximately 1 copy per chromosome) renders the host resistant to both kanamycin and tetracycline (KMrTcr) . Three plasmids were not self-transmissible . The restriction endonuclease cleavage maps of the two plasmids, pTB19 and pTB20, were constructed . pTB19 and pTB20, both of which were originally isolated from thermophilic bacilli, were tested for stability in B . subtilis . Digestion of pTB19 followed by ligation yielded deletion plasmids pTB512 (Kmr), pTB52 (Tcr), and pTB53 (KmrTcr) . Determinants of Kmr, Tcr, and DNA replication were associated with EcoRI fragments R1b (4.2 X 10(6) daltons), R3 (2.8 X 10(6) daltons), and R1a (4.2 X 10(6) daltons), respectively . Restriction endonuclease cleavage maps of pTB51, pTB52, and pTB53 were constructed . Tetracycline resistance of pTB20 was confirmed to be in the EcoRI fragment (1.85 X 10(6) daltons).

Biochim Biophys Acta, 1981 May 13, 635(3), 476 - 87
The effect on photosynthetic electron transport of temperature-dependent changes in the fluidity of the thylakoid membrane in a thermophilic blue-green alga; Hirano M et al.; Various electron transport reactions in cell or isolated thylakoid membranes of the thermophilic blue-green alga, Synechococcus sp . were measured at different temperatures between 72 and 3 degrees C . They are classified into two groups with respect to their temperature dependency . The first group involves cytochrome 553 photooxidation, methyl viologen photoreduction with reduced 2,6-dichlorophenolindophenol as electron donor and 3-(3',4'-dichlorophenyl)-1,1-dimethylurea-resistant ferricyanide photoreduction determined in the presence or absence of silicomolybdate . The Arrhenius plot of these reactions showed a single straight line with the activation energy of about 10 kcal/mol throughout wide temperature ranges studied . Methyl viologen photoreduction with water as electron donor, reduction of flash-oxidized cytochrome 553, ferricyanide photoreduction and photosynthetic O2 evolution form the second group . Their arrhenius plots are characterized by discontinuities or breaks at about 30 and 10 degrees C, which respectively correspond to the upper and lower boundaries of the lateral phase separation of the membrane lipids . The first group reactions represent short spans of electron transport which are mediated either by Photosystem I or Photosystem II alone and not related to plastoquinone, whereas all the reactions of the second group involve plastoquinone . It is concluded therefore that the membrane fluidity affect electron transport specifically at the region of plastoquinone . It is proposed that the reaction center chlorophyll-protein complexes of both Photosystems I and II are closely associated with related electron carrier proteins to form functional supramolecular assemblies so that electron transfer within such a cluster of proteins proceeds independently of the phase changes in the membrane lipids . On the other hand, the role of plastoquinone as a mobile electron carrier mediating electron transfer from the protein assembly of Photosystem II to that of Photosystem I through the fluid hydrophobic matrix of the membranes is highly sensitive to the physical state of the membrane lipids.

Res Commun Chem Pathol Pharmacol, 1981 May, 32(2), 329 - 34
Attempts at induction of phenylalanine hydroxylase in thermophilic bacteria and induction of thermophily in mesophiles; Weiss B et al.; Some moderate and extreme thermophilic bacteria grew well on media other than the recommended basic media . Attempts to induce phenylalanine hydroxylase (Phe H) in the various thermophiles as well as mild thermophily in the mesophiles Pseudomonas sp ATCC 11299a and Chromobacterium violaceum ATCC 12540 were unsuccessful . Evidence is presented indicating that the enzyme in the latter two organisms may be membrane bound . The level of Phe H activity induced was not always consistent with the level of the inducer phenylalanine (Phe) in the growth medium.

J Biochem (Tokyo), 1981 May, 89(5), 1501 - 11
The mechanism of preferential synthesis of poly{r(purine)} in the transcription of poly{d(purine)} . poly{d(pyrimidine)} by T . thermophilus RNA polymerase; Tsuji S et al.; The mechanism of preferential transcription on poly{d(purine)} . poly{d(pyrimidine)} was investigated using RNA polymerase of T . thermophilus HB8 . Though the machinery for initiation is lacking, the core enzyme has the latent ability to synthesize poly{r(pyrimidine)} as well as poly{r(purine)} . The holoenzyme can synthesize poly{r(purine)} in the usual manner . Poly{r(pyrimidine)} synthesis by the holoenzyme is, however, forbidden . These results suggest that the sigma factor plays a crucial role in this preferential transcription, and that this preferential transcription may be useful as a model for the sense strand recognition . Various results led us to the hypothesis that the high affinity site for the poly{d(pyrimidine)} strands on the enzyme plays a very important role.

J Cell Biol, 1981 May, 89(2), 373 - 8
Decoration of spindle microtubules with Dynein: evidence for uniform polarity; Telzer BR et al.; Studies were conducted to determine whether the microtubules present within native spindles isolated from eggs of the surf clam, Spisula solidissima, could bind dynein obtained from axonemes of Tetrahymena thermophila . SDS gel electrophoresis revealed that the high molecular weight polypeptides that make up dynein cosedimented with the isolated spindles . Moreover, the ATPase activity of dynein bound to the spindle microtubules was stimulated approximately sevenfold . The birefringence retardation of spindles incubated without dynein decreased from 1.4 nm to an undetectable level within 45 min, whereas that of spindles incubated for the same period of time with dynein was 1.0 nm, approximately 70% of its initial value, thereby indicating that dynein stabilized spindle birefringence . Ultrastructural analysis revealed that each spindle microtubule was decorated with four to seven dynein arms attached by their "B" end, that which cross-bridges the B-subfiber within native axonemes . In addition, the polarity of the spindle microtubules could be determined by the orientation of the bound dynein arms . The results of these studies suggest that the half-spindle is composed of microtubules possessing the same polarity.

Biochim Biophys Acta, 1981 Apr 28, 668(2), 277 - 81
Purification, some properties and amino acid sequence of Thermus thermophilus HB8 ferredoxin; Sato S et al.; A stable ferredoxin was purified in a crystalline form from an aerobic, thermophilic bacterium, Thermus thermophilus HB8 . The molecular weight of the protein was determined to be 10500 by gel-filtration on Sephadex G-75 and to be 10200 by the sedimentation equilibrium method . The number of iron and acid labile sulfur atoms per mol was determined to be 6.3 and 6.4, respectively . The optical absorption spectrum of the ferredoxin has a broad maximum around 400 nm . The ferredoxin was so thermostable that its absorbance at 400 nm did not decrease after a 45-min incubation at 65 degrees C . The primary structure of the ferredoxin consisting of 78 amino acids was determined by sequence analysis of peptides obtained from a tryptic digest of the S-carboxymethylated ferredoxin and from a Staphylococcus aureus V8 protease digest of the S-aminoethylated derivative . The distribution of cysteine residues and the amino acid sequence around the cysteine residues are very similar to those of Mycobacterium smegmatis ferredoxin.

J Biol Chem, 1981 Apr 25, 256(8), 3612 - 4
Kinetic changes in protein synthesis in response to a sublethal heat shock in starved Tetrahymena thermophila; Hauser L et al.; We have analyzed the alterations that occur in the patterns of protein synthesis of Tetrahymena thermophila cells when these cells are exposed to a sublethal heat treatment . At least 11 major polypeptides, ranging in size from 97,000 to 27,000 daltons, are produced when the incubation temperature of Tetrahymena cultures is raised from 29 to 41 degrees C . The kinetics of appearance and disappearance appears to differ for each one of these heat-induced polypeptides, suggesting that elaborate mechanisms might operate in the regulation of the expression of this group of genes.

Can J Microbiol, 1981 Apr, 27(4), 444 - 51
K+, Na+, and Mg2+ content and permeability of Methanospirillum hungatei and Methanobacterium thermoautotrophicum; Sprott GD et al.; The K+, Na+, and Mg2+ contents of Methanospirillum hungatei and of the thermophile Methanobacterium thermoautotrophicum were determined at various phases of growth . The intracellular K+ content of exponential phase cells of M . thermoautotrophicum (approximately 780 mM) was 5.4-fold higher than in M . hungatei, and decreased gradually as the culture entered the stationary phase . Both methanogens concentrated Mg2+, exhibiting an increased content as the cultures aged . Comparisons among extraction methods showed that most of the internal K+ was readily released, but a minimum of half of the Mg2+ in M . hungatei, and most of the M2+ in M . thermoautotrophicum, was in a bound form . Exponential phase of cells of M . hungatei established an intracellular level of Na+ lower than the outside medium, but the thermophile concentrated Na+ . Dextran, inulin, sucrose, and glucose penetrated cell pellets to varying degrees and could be used to measure the space corresponding to cytoplasm and to cell wall permeability barriers . L-Phenylalanine penetrated fully and acetate accumulated in both methanogens . Acetate uptake in cell suspensions of M . hungatei was fully inhibited by oxygen . N-ethylmaleimide, or N,N'-dicyclohexylcarbodiimide, but was not affected by the proton conductor carbonylcyanide p-trifluoromethoxyphenyl-hydrazone . L-Malate, which penetrated M . hungatei cells poorly, was metabolized to glutamate, indicating the presence of an incomplete reductive carboxylic acid cycle.

J Bacteriol, 1981 Apr, 146(1), 192 - 9
Ethanol production by thermophilic bacteria: metabolic control of end product formation in Thermoanaerobium brockii; Ben-Bassat A et al.; Specific changes in the chemical and microbial composition of Thermoanaerobium brockii fermentations were compared and related to alterations of process rates, end product yields, and growth parameters . Fermentation of starch as compared with glucose was associated with significant decreases in growth rate and intracellular fructose-1,6-bisphosphate concentration and with a dramatic increase in the ethanol/lactate product ratio . Glucose or pyruvate fermentation in the presence of acetone was correlated with increased substrate consumption, growth (both rate and yield), acetate yield, and quantitative reduction of acetone to isopropanol in lieu of normal reduced fermentation products (i.e., H2, ethanol, lactate) . Acetone altered pyruvate phosphoroclastic activity of cell extracts in that H2, lactate, and ethanol levels decreased, whereas the acetate concentration increased . Glucose fermentation in the presence of exogenous hydrogen was associated with inhibition of endogenous H2 production and either increased ethanol/acetate product ratios and decreased growth at less than 0.5 atm (51 kPa) of H2 or total growth inhibition at 1.0 atm (102 kPA) . The effects of exogenous hydrogen on glucose fermentation were totally reversed by the addition of acetone . Glucose fermentation in coculture with Methanobacterium thermoautotrophicum correlated with increased growth (both rate and yield), acetate yield, and the formation of methane in lieu of monoculture reduced products . In coculture, but not monoculture, T . brockii grew on ethanol as the energy source, and acetate and methane were the end products as a direct consequence of hydrogen consumption by the methanogen.

Biochem J, 1981 Apr 1, 195(1), 183 - 90
Novel NADP-linked alcohol--aldehyde/ketone oxidoreductase in thermophilic ethanologenic bacteria; Lamed RJ et al.; An NADP-specific alcohol--aldehyde/ketone oxidoreductase was detected in cell extracts of Thermoanaerobium brockii and Clostridium thermohydrosulfuricum, but not in Thermobacteroides acetoethylicus or Clostridium thermocellum . The enzyme was purified from Ta . brockii by differential procedures that included heat treatment and an affinity-chromatography step on Blue Dextran--Sepharose . The 44-fold-purified enzyme displayed one band (mol.wt . approx . 40000) after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis . The enzyme had a broad substrate specificity that included linear and branched primary alcohols, linear and cyclic secondary alcohols, linear and cyclic ketones, and acetaldehyde . The NADP-specific alcohol--aldehyde/ketone oxidoreductase was considerably more active towards secondary alcohols than towards other substrates . The enzyme had remarkable stability to heating at 86 degrees C for 70 min, but was rapidly denatured on boiling . Secondary-alcohol dehydrogenase activity displayed a noticeable inflexion point at 50 degrees C in Arrhenius plots and a high Q10 value (greater than 2.0) . The enzyme was inactivated by the thiol-blocking reagent p-chloromercuribenzoate, but was not significantly inhibited by common metal-ion-binding agents . The NADP-linked alcohol--aldehyde/ketone oxidoreductase of Ta . brockii appears to have properties distinct from those of previously described primary- and secondary-alcohol dehydrogenases.

Gene, 1981 Apr, 13(3), 281 - 7
Extrachromosomal rDNA of Tetrahymena thermophila is not a perfect palindrome; Kiss GB et al.; We have determined the restriction-endonuclease-cleavage map and the nucleotide sequence of the central 1.4 kb fragment of the macronuclear extrachromosomal rDNA of Tetrahymena thermophila . These data demonstrate that this molecule is not a perfect palindrome, having a 29 bp AT-rich non-palindromic sequence at its center . This observation is important in determining the mechanism by which a single chromosomally integrated rRNA gene in the micronucleus is rearranged and amplified during sexual development to yield multiple copies of extrachromosomal rDNA in the macronucleus.

Nucleic Acids Res, 1981 Mar 25, 9(6), 1451 - 61
Phylogenetic tree derived from bacterial, cytosol and organelle 5S rRNA sequences; Kuntzel H et al.; A phylogenetic tree was constructed by computer analysis of 47 completely determined 5S rRNA sequences . The wheat mitochondrial sequence is significantly more related to prokaryotic than to eukaryotic sequences, and its affinity to that of the thermophilic Gram-negative bacterium Thermus aquaticus is comparable to the affinity between Anacystis nidulans and chloroplastic sequences . This strongly supports the idea of an endosymbiotic origin of plant mitochondria . A comparison of the plant cytosol and chloroplast sub-trees suggests a similar rate of nucleotide substitution in nuclear genes and chloroplastic genes . Other features of the tree are a common precursor of protozoa and metazoa, which appears to be more related to the fungal than to the plant protosequence, and an early divergence of the archebacterial sequence (Halobacterium cutirubrum) from the prokaryotic branch.

J Biol Chem, 1981 Mar 25, 256(6), 2873 - 7
pH dependence of H+ conduction through the membrane moiety of the H+-ATPase (F0 . F1) and effects of tyrosyl residue modification; Sone N et al.; A convenient and reliable method to measure passive H+-translocating activity (H+ conductivity) was developed; vesicles reconstituted from the membrane moiety (F0) of H+-ATPase (F0 . F1) and soybean phospholipids were loaded with KCl by a freeze-thaw-sonication procedure and the rate of H+ uptake caused by the K+ diffusion potential upon addition of valinomycin was followed with a pH meter . Of the methods tested, a dialysis method using cholate plus deoxycholate gave the best results for reconstitution . Using this method, H+ conductivity of the membrane moiety of H+-ATPase from a thermophilic bacterium PS3 (TF0) was analyzed . Dependence of H+ conductivity of TF0 on H+ concentration fitted a Michaelis-Menten equation showing a Vmax of 31.3 microgram ion/min . mg of TF0 and a Km of 0.095 microgram ion/liter . Upon modification of a tyrosyl residue of TF0 with iodine, the Km value shifted to 0.71 microgram ion/liter, while the Vmax remained constant . These results were interpreted as indicating that a single tyrosyl residue in N,N'-dicyclohexylcarbodiimide-binding proteolipid of TF0 plays an important role as an H+ donor in the the rate-limiting step of H+ permeation through TF0 . TF1, the catalytic moiety of H+-ATPase from the thermophilic bacterium PS3, blocked H+ conduction through TF0 . A 1:1 stoichiometry of TF1 and TF0 was found in ATP-dependent membrane potential generation as well as H+ conduction.

Biochem J, 1981 Mar 15, 194(3), 877 - 87
Properties of oxygen-evolving photosystem-II particles from Phormidium laminosum, a thermophilic blue--green alga; Stewart AC et al.; 1 . O2-evolving Photosystem-II particles from the thermophilic blue--green alga Phormidium laminosum contained 1 mol of Mn/13--17 mol of chlorophyll a compared with 1 mol of Mn/65--75 mol of chlorophyll a in unfractionated membranes . 2 . At least two-thirds of the Mn in the Photosystem-II particles was removed by mild heating and by treatment with Tris or EDTA, with concomitant loss of O2 evolution . However, irreversible inactivation was also caused by washing in buffers without MgCl2, and this inactivation was not accompanied by a corresponding loss of Mn . 3 . Bivalent cations (Mg2+ or Ca2+), Cl- or Br- ions and at least 20% (v/v) glycerol were required for maximum stability of O2 evolution . 4 . The Photosystem-II particles were enriched in high-potential cytochrome b-559 (1 mol of cytochrome/50--60 mol of chlorophyll a) and in component C-550, and had a photosynthetic-unit size of 40--70 molecules of chlorophyll a . 5 . The absorption spectrum at 77 K showed a preponderance of shorter-wavelength forms of chlorophyll a in the Photosystem-II particles, and in the fluorescence emission spectrum at 77 K there were major chlorophyll fluorescence bands at 684 nm and 695 nm, with almost no fluorescence in the far-red region . 6 . Analysis of the lipid and protein contents showed that the Photosystem-II particles were not chemically pure (for example, all of the membrane-bound cytochromes and cytochrome c-549 were present), but their high O2-evolution activity and good optical properties make them useful for functional studies on Photosystem-II and O2 evolution.

Mech Ageing Dev, 1981 Mar, 15(3), 235 - 8
Age-associated changes in the micronuclear cycle of Tetrahymena thermophila A III heterokaryons . A brief note; Shabatura SK et al.; The micronuclear cycle of Tetrahymena thermophila A III heterokaryons is shown to change with increasing clonal age . Autoradiographic and cytofluorimetric studies suggest that alteration may be due to (1) loss of late replicating sequences, or (2) (more likely) changes in the timing of micronuclear division and S with respect to cytokinesis.

Mikrobiologiia, 1981 Mar-Apr, 50(2), 268 - 71
{Ultrastructural organizational characteristics of the hydrogen thermophilic bacterium, Pseudomonas thermophila}; Kostrikina NA et al.; The ultrastructural organization of the lithotrophic hydrogen bacterium Pseudomonas thermophila K2 was described for the first time and was found to be typical of Gram-negative bacteria . The ultrastructure of this organism is characterized by (i) irregular plication of the outer membrane of the cell wall and a very thin (2-3 nm) rigid layer; (ii) a considerable number of intracellular membranes differing in their structure and location; (iii) fragmentation of the cytoplasm involving the plasmalemma and the cell wall; (iv) the presence, in the nucleoid zone, of paracrystals having a hexagonal shape and resembling carboxysomes in their size, shape and macromolecular organization, which had not been found in hydrogen bacteria hitherto.

Arch Microbiol, 1981 Mar, 129(1), 29 - 31
The stringent response to unacylated tRNA, energy-and temperature-downshift in Bacillus stearothermophilus; Fehr S et al.; The response of the thermophile Bacillus stearothermophilus to inhibition of tRNA acylation, energy starvation and temperature downshift was characterized . We found that B . stearothermophilus, like other prokaryotic organisms, reacts with the so-called stringent response, which includes the accumulation of the unusual nucleotides guanosine 3',5' bis (diphosphate) {ppGpp} and guanosine 3'-diphosphate, 5'-tri-phosphate {pppGpp} and concomitantly the reduction of RNA synthesis and growth rate . The amount of (p)ppGpp formed depended on the cause of the stringent response: when tRNA acylation was inhibited (p)ppGpp synthesis was much higher than after energy starvation or temperature downshift whereas RNA synthesis was totally blocked in each case.

J Biochem (Tokyo), 1981 Feb, 89(2), 677 - 82
Cloning of 3-isopropylmalate dehydrogenase gene of an extreme thermophile and partial purification of the gene product; Tanaka T et al.; The gene of an extreme thermophile, Thermus thermophilus HB8, which codes for a leucine biosynthetic enzyme, 3-isopropylmalate (3-IPM) dehydrogenase {EC 1.1.1.85}, was cloned in Escherichia coli using pBR322 as a vector . E . coli cells carrying this recombinant plasmid, pHB2, produced the thermophilic enzyme 7-fold more than did T . thermophilus HB8 cells . When the crude extract of the pHB2-carrying cells was treated at 70 degrees C for 10 min, approximately 75% of the protein in the extract was precipitated with full activity of the thermophilic 3-IPM dehydrogenase being left in the supernatant, indicating that 4-fold purification was achieved during this process . This shows that the thermophilic 3-IPM dehydrogenase was purified 28-fold by these two procedures, cloning and heat treatment, and demonstrates that the extract from the plasmid-harboring cells is a good starting material for purification of the enzyme . Following the heat treatment, 3-IPM dehydrogenase was further purified by ammonium sulfate precipitation and DEAE-cellulose column chromatography . The enzyme preparation thus obtained contained 3-IPM dehydrogenase as a major component with a few minor impurities as shown by polyacrylamide gel electrophoresis, whereas the enzyme preparation from T . thermophilus HB8 cells obtained by the same procedures showed multiple bands on a polyacrylamide gel electrophoresis.

J Dairy Res, 1981 Feb, 48(1), 139 - 48
Effect of the addition of extracts of thermophilic lactobacilli on acid production by Streptococcus thermophilus in milk; Hemme DH et al.; Soluble extracts of 20 strains of thermophilic lactobacilli (Lactobacillus helveticus, L . lactis and L . bulgaricus) were prepared and added to milk for the culture of 10 strains of Streptococcus thermophilus . Acid production was stimulated in 64.5% of cases for 9 of these 10 strains . The L . helveticus extracts were the most stimulatory, but the same extracts did not always strongly stimulate each strain of Str . thermophilus . The stimulatory effects observed varied with the volume of extract and the strain of Str . thermophilus . The exception was Str . thermophilus 385, which was never stimulated . The stimulatory effects observed were due to aminopeptidases present in the lactobacillus extracts and were not related to a general caseinolytic activity . The possible addition of such extracts to milk for cooked hard cheese is discussed.

Biochem J, 1981 Feb 1, 193(2), 379 - 87
Purification and characterization of a thermostable glucoamylase from the thermophilic fungus Thermomyces lanuginosus; Basaveswara Rao V et al.; Glucoamylase (1,4-alpha-D-glucan glucohydrolase, EC 3.2.1.3) was purified from the culture filtrates of the thermophilic fungus Thermomyces lanuginosus and was established to be homogeneous by a number of criteria . The enzyme was a glycoprotein with an average molecular weight of about 57 000 and a carbohydrate content of 10-12% . The enzyme hydrolysed successive glucose residues from the non-reducing ends of the starch molecule . It did not exhibit any glucosyltransferase activity . The enzyme appeared to hydrolyse maltotriose by the multi-chain mechanism . The enzyme was unable to hydrolyse 1,6-alpha-D-glucosidic linkages of isomaltose and dextran . It was optimally active at 70 degrees C . The enzyme exhibited increase in the Vmax . and decreased in Km values with increasing chain length of the substrate molecule . The enzyme was inhibited by the substrate analogue D-glucono-delta-lactone in a non-competitive manner . The enzyme inhibited remarkable resistance towards chemical and thermal denaturation.

Cell, 1981 Feb, 23(2), 467 - 76
The intervening sequence of the ribosomal RNA precursor is converted to a circular RNA in isolated nuclei of Tetrahymena; Grabowski PJ et al.; The Tetrahymena thermophila ribosomal RNA gene contains an intervening sequence (IVS), which is transcribed as part of the precursor RNA and subsequently removed by splicing . We have found previously that the IVS is excised as a 0.4 kb RNA in isolated nuclei . We now report the finding of a novel RNA molecule, which is an electrophoretic variant (EV) of this 0.4 kb IVS RNA . The EV was identified as a form of the IVS RNA by Southern hybridization, RNA fingerprinting and R-loop mapping . A pulse-chase experiment established that in vitro the excised IVS RNA is converted to the EV by a post-splicing event . This conversion is enhanced at 39 degrees C compared to 30 degrees C and is irreversible under our experimental conditions . The EV of the IVS is a circular RNA . This structure was first suggested by its anomalous electrophoretic mobility on denaturing compared to nondenaturing gels . When the EV was prepared for electron microscopy under totally denaturing conditions, 0.4 kb circular molecules were observed . Furthermore, we have converted the circular form to a linear form by limited T1 RNAase digestion . The circular RNA survived treatment with DNAase, protease, glyoxal and various denaturants, which suggests that it is a covalently closed RNA circle.

J Protozool, 1981 Feb, 28(1), 127 - 31
Isolation of a stearoyl CoA Desaturase form Tetrahymena thermophila; Bertram J et al.; Cell free preparations of Tetrahymena thermophila contain an enzyme that catalyzes the direct desaturation of stearoyl CoA to octadecenoic acid . The enzyme is associated with the microsomal fraction of the ciliate . Substrate of the enzyme consists of either free stearic acid or stearoyl CoA . Both ATP and CoA are required when free stearate is the substrate and are also highly stimulatory when stearoyl CoA is the substrate . With stearoyl CoA as the substrate, either NADH or NADPH are required for desaturase activity . In presence of ATP and CoA, either NAD or NADP can replace NADH and NADPH . Desaturase activity is optimal when the enzyme is incubated at pH of 7.2 and a temperature of 30-35 degrees C . Highest levels of the stearoyl CoA desaturase are found in stationary phase ciliates grown at 35 degrees C.

Biochim Biophys Acta, 1981 Jan 14, 634(1), 93 - 104
Comparison of the redox reactions of various types of cytochrome c with iron hexacyanides; Kihara H; The dynamic behavior of various types of cytochromes c in the redox reaction with iron hexacyanides was studied using a temperature-jump method in order to elucidate the molecular mechanism of the redox reaction of cytochromes with their oxidoreductants . Transmittance after the temperature jump changed through a single exponential decay for all cytochromes investigated . Under a constant concentration of anion, the redox reaction of various types of cytochrome c with iron hexacyanides was analyzed according to the scheme: (see formula in text) where C(III) and C(II) are ferric and ferrous cytochromes, respectively, Fe(III) and Fe(II) are ferri- and ferrocyanides, respectively, C(III) . Fe(II) is the ferricytochrome-ferrocyanide complex and C(II) . Fe(III) is the ferrocytochrome-ferricyanide complex . When step B is slower than the other two steps A and C, tau-1 can be represented approximately as (see formula in text) where the bar over the variables denotes the equilibrium value . In a large excess of ferrocyanide against cytochrome, we can estimate kappa 2, kappa-2, K1 and K3 independently . In the case of horse cytochrome c at 18 degrees C in 0.1 M phosphate buffer at pH 7 with 0.3 M KNO3, the estimated parameters are kappa 2 = 100 +/- 50 S-1, kappa-2 = (3.5 +/- 1.0) . 10(3) S-1, K1 = 15 +/- 7 M-1 and K3 = (8.5 +/- 1.5) . 10(-4) M . From the same experiments for seven cytochromes (cytochrome c from horse, tuna, Candida krusei, Saccharomyces oviformis, Rhodospirillum rubrum cytochrome c2, Spirulina platensis cytochrome c-554 and Thermus thermophilus cytochrome c-552), the following results can be deduced . (1) Each parameter defined in the scheme above (kappa 2, kappa-2, K1, K3) diverged beyond the error range . Above all, kappa 2 values of cytochromes c-554 and c-552 are as large as 1 . 10(4) S-1 and much larger than those for the other cytochromes (to 50 approx . 700 S-1) . (2) The variance of kappa 2K1 and kappa-2/K3 are relatively less than the variances of individual parameters (kappa 2, kappa-2, K1 and K3), which suggests that the values of kappa 2K1 and kappa-2/K3 have been conserved during the course of evolution.

J Biol Chem, 1981 Jan 10, 256(1), 148 - 53
Identification of an essential glutamic acid residue in the beta subunit of the adenosine triphosphatase from the thermophilic bacterium PS3; Yoshida M et al.; The TF1-ATPase from the thermophilic bacterium, PS3, is inactivated by dicyclohexylcarbodiimide (DCCD) . This inactivation is stimulated by ADP and other specific nucleotides and is inhibited by Mg2+ . When the inactivation is carried out with {14C}DCCD, about 2 g atoms of 14C are bound/mol of TF1 when the enzyme is nearly completely inactivated . The isolated subunits from TF1 inactivated with {14C}DCCD contain the following amounts of 14C/mol: alpha, 0.12 g atom; beta, 0.47 g atom; gamma, approximately 0.04 g atom; delta, none; and epsilon, 0.05 g atom . Fractionation of tryptic digests have shown that the 14C bound to the alpha subunit is nonspecifically associated with several peptides, and that the 14C bound to the beta subunit is associated with a single tryptic peptide with the amino acid sequence Ala-Gly-Val-Gly-Glu-Arg, where Glu represents the N-gamma-glutamyl derivative of dicyclohexyl{14C}urea.

C R Seances Acad Sci III, 1981 Jan 5, 292(1), 41 - 3
{Isolation of 2 thermophilic methanogenic strains belonging to the Methanobacterium family}; Marty DG et al.; Two thermophilic methanogenic bacteria were respectively isolated from bovine and swine wastes . Morphologically, these two strains belong to the genus Methanobacterium, but by their nutritional properties (growth factor requirements and formate utilization as methanogenic substrate), they seem to be different from Methanobacterium thermoautotrophicum.

J Bacteriol, 1981 Jan, 145(1), 503 - 12
Sequence homology in the amino-terminal and active-site regions of thermolabile glyceraldehyde-3-phosphate dehydrogenase from a thermophile; Crabb JW et al.; The unusual thermolability of glyceraldehyde-3-phosphate dehydrogenase from the facultative thermophile Bacillus coagulans KU (Crabb et al., Biochemistry 16:4840-4847, 1977) has provided the first opportunity to study a homologous enzyme from the same genus that exhibits a marked difference in thermostability . In pursuit of the structural bases for the thermostability of proteins, the sequences of the amino terminus (residues 1 through 27) and the active-site cysteine cyanogen bromide peptide (residues 130 through 167) of this enzyme have been determined and compared with sequences of the enzyme from other sources . The importance of comparing phylogenetically related proteins is evident from the 87% identity found between these sequences in the enzyme from B . coagulans and Bacillus stearothermophilus, versus only 45% identity for all other known sequences . The marked sequence identity of the enzyme from the two Bacillus species drew attention to the variable region (residues 138 through 140a) which is exposed to the exterior of the quaternary structure of this enzyme . Based on the reported crystallographic structures of the enzyme from lobster muscle and B . stearothermophilus and space-filling models of the variable region, the segment Asp-Pro-Lys-Ala in B . stearothermophilus should be more thermostable than the analogous sequence, Asp-Ala-Ala-Asn, from B . coagulans . In addition, the space-filling models suggested that the spatial relationship of an amino acid side chain and its potential for close packing and interactions with neighboring side chains may be more important than the type of amino acid substituted.

Folia Microbiol (Praha), 1981, 26(5), 364 - 9
Bacillus stearothermophilus from Saudi Arabian Soils; Abu-Zinada AH et al.; Ten thermophilic Gram-positive bacteria were isolated from various soils of Saudi Arabia . The strains are spore-forming rods belonging to the species Bacillus stearothermophilus . The cells are motile, strictly aerobic, catalase and oxidase positive . The sporangia appear to be swollen and their position varies from terminal in some to sub-terminal in others . The thermal stability of some enzymes of these bacteria was investigated; extracellular alpha-amylase appears to be very sensitive to pH and temperature . The ultrastructure of these bacteria shows specific changes in the cell wall when grown at the maximum, minimum and optimum growth temperatures, respectively.

Basic Life Sci, 1981, 18, 441 - 61
Thermophilic ethanol fermentations; Zeikus JG et al.; Thermophilic ethanol fermentations are of interest to industrial alcohol production because both the pentose and hexose fraction of biomass can be directly fermented in high yield (i.e., mol ethanol/mol substrate consumed), and because of potential novel process features associated with high temperature operation . As a net result, the co-culture cellulose fermentations described here may have the potential to convert more substrate to alcohol than some other bioconversion systems described {see Figure 11, (2)} . However, considerably more fundamental and applied research is required before realistic economic assessments can be made . Detailed analysis of the data presented above suggests key control parameters for thermophilic ethanol production (see Table IX) . Understanding in detail the physiological and biochemical features that control rate limitation, yield limitation and concentration limitation appears to me as trends for future applied and fundamental studies on thermophilic ethanologenic bacteria . It is worth noting from the data reviewed here that understanding control of any one of these 3 major limitations is complex and multi-faceted . Indeed, improvement of ethanol tolerance (i.e . the ability to produce greater than 1% ethanol at high rates) in these bacteria appears to involve challenges by all three limitations . Furthermore, the biochemical basis for alcohol tolerance in thermophilic ethanologens appears to vary in different species . For example, the ethanol dehydrogenase of C . thermocellum is inhibited by physiological concentrations of alcohol (i.e . 1%) whereas, the reversible activity of T . brockii or C . thermohydrosulfuricum enzyme is increased by higher solvent concentration (greater than 5%).

Basic Life Sci, 1981, 18, 397 - 419
Some aspects of thermophilic and extreme thermophilic anaerobic microorganisms; Ljungdahl LG et al.; In this presentation, we have discussed that the acetogenic thermophilic bacterium, Clostridium thermoaceticum, ferments glucose almost quantitatively to acetate . That part of the acetate is formed from CO2, which functions as the electron sink . We have demonstrated that enzymes in the acetate formation contain trace elements such as iron, cobalt, nickel, selenium and tungsten . Furthermore, we have indicated that this bacterium must have an electron transport system, which is not yet completely understood . With Clostridium thermohydrosulfuricum we have obtained results which indicate that this thermophile may selectively produce proteins dependent on the environmental temperature . We have presented a new bacterium, Thermoanaerobacter ethanolicus, which ferments several sugars including starch, cellobiose, and xylose to ethanol . We have demonstrated the existence in a thermal environment of anaerobic bacteria that grow at temperatures of around 90 degrees C and which are capable of fermenting diverse substrates such as lactate, glucose, and cellulose.

Folia Microbiol (Praha), 1981, 26(2), 89 - 94
Transport properties of two extremely thermophilic species of Thermus; Michaljanicova D et al.; Thermus flavus and T . ruber grew optimally at 75 and 60 degrees C, respectively, but transport of monosaccharides (D-quinovose) and amino acids (2-aminoisobutyric acid) had optima about 20 degree C lower . Both transports were active, inhibited by 2,4-dinitrophenol but hardly at all by uranyl(2+) ions . Several transport systems are apparently involved with each class of compounds . Preincubation with glucose curtailed subsequent transport severely . Practical cessation of transport below 35 degrees C may be associated with the rather uniform composition of membrane lipids where iso- and anteiso-C15 and C17 acids are practically the only components.

Mikrobiologiia, 1981 Jan-Feb, 50(1), 49 - 54
{Growth of obligate-thermophilic bacteria on a medium with paraffin}; Loginova LG et al.; The obligate thermophilic bacteria Bacillus stearothermophilus (the optimal growth temperature 55-73 degrees C) and Thermus ruber (the optimal growth temperature 60 degrees C) were isolated from hot water springs of the Kunashir Island for the first time in a liquid mineral medium with paraffin . Some of the bacteria belonging to Bac . stearothermophilus grew at a high rate in a liquid medium with paraffin (strain 16); other strains (12a, 12b) were capable of growing only together with Thermus ruber; some strains could grow only on a solid medium with paraffin (strains 14a, 14b).

Zentralbl Bakteriol Naturwiss, 1981, 136(8), 661 - 81
{Enzymatic studies of the thermophilic hydrocarbon utilizing fungi strains Aspergillus fumigatus and Mucor lusitanicus (author's transl)}; Bemmann W et al.; The activities of catalase, peroxydase, and of the chitinolytic enzymes of the thermophilic hydrocarbon-utilizing fungal strains of Aspergillus fumigatus and Mucor lusitanicus, grown in n-alkane (KW) or glucose (Glc) medium at different temperatures with additions of KCN, NaNO3, 3-amino-1,2,4-triazole (ATA), and CaCl2 and with a shift culture from Glc to KW medium were determined . The enzymatic activities, able to cleave H2O2, were cytochemically shown in the hyphae with 3,3-diamino-benzidine-tetrahydrochloride (DAB) reagent . The colouring of the mycelium was most intense during the linear growth phase in the KW medium . The DAB oxydation could completely by suppressed by pre-incubation of the mycelium with the enzyme inhibitors KCN or ATA and incubation in the standard medium without H2O2 . The catalase and peroxydase activities were higher in the KW medium than in the Glc medium, where highest activities occurred at the start of the linear and the stationary growth phase . The pre-incubation of the enzyme solution with the inhibitors KCN, NaNO2 or ATA gave maximum inhibition with ATA, likewise gave the addition of ATA to the medium the highest inhibition of the enzymatic activities, connected with an extension of the initial growth phase . Addition of CaCl2 increased the catalase and peroxydase activities, where catalase at 40 degrees C and peroxydase at 50 degrees C showed maximum growth . A shift of the growing mycelium from Glc to KW medium confirmed the correlation of KW utilization and high catalase activity . The highest chitinolytic activities were ascertained at the beginning of the linear growth phase at a temperature of 40 degrees C . The results were discussed.

Zentralbl Bakteriol Naturwiss, 1981, 136(7), 590 - 602
{The growth of thermophilic fungi strains Aspergillus fumigatus and Mucor lusitanicus in n-alkane medium (author's transl)}; Voigt A et al.; Growth parameters of the thermophilic hydrocarbon utilizing strains Aspergillus fumigatus and Mucor lusitanicus in n-alkanes (KW)- and glucose (GIc)-medium in standing and fermenter cultures were investigated . The temperature limits for growth were dependent on the medium composition . The temperature range for sporulation was smaller than for the hyphal growth . Both strains were cultivated on carbon limitation with subsequent determinations of n-alkanes glucose, ammoniumsulfate-nitrogen, phosphate-phosphorus and the trace elements Mn, Cu, Zn and Fe . A method for estimation of hydrocarbon utilization was developed . In a medium containing a mixture of n-alkanes and glucose as C-sources the strains utilized at first glucose . Both the strains excreted monocarbon acids with a low carbon number into the culture liquid . The results were discussed.

Mikrobiologiia, 1981 Jan-Feb, 50(1), 13 - 20
{Electron transport chain in a thermophilic methane-oxidizing culture of Methylococcus thermophilus}; Sokolova IG et al.; The electron transport chain was studied in the obligate methane oxidizing culture of Methylococcus thermophilus during the oxidation of methanol (the source of carbon) which is an oxidized derivative of methane as well as during the oxidation of hydroxylamine which is an intermediate in the oxidation of ammonium (the source of nitrogen) by Mc . thermophilus cells . Cytochromes a, b and c are involved in electron transport . Cytochrome cco and cytochrome c554 have been isolated from the cell-free extract of Mc . thermophilus and purified . A scheme for electron transport operating in the oxidation of methanol and hydroxylamine is suggested on the basis of studying the characteristics of these cytochromes . Cytochrome a was shown to be a component of terminal oxidase . Cytochromes b are connected with membranes and also found in the composition of hydroxylamine oxidase . Cytochrome cco and, possibly, terminal oxidase (cytochromes a) are involved, in the oxidation of CH3OH by methanol dehydrogenase, in electron transport; cytochrome c554 as well as cytochrome b and c in the composition of hydroxylamine oxidase participate in electron transport in the oxidation of NH2OH by hydroxylamine oxidase . The characteristics of the electron transport system in Mc . thermophilus are discussed.

Biochem J, 1981 Jan 1, 193(1), 67 - 74
Substrate specificity and mode of action of the cellulases from the thermophilic fungus Thermoascus aurantiacus; Shepherd MG et al.; The substrate specificities of three cellulases and a beta-glucosidase purified from Thermoascus aurantiacus were examined . All three cellulases partially degraded native cellulose . Cellulase I, but not cellulase II and cellulase III, readily hydrolyzed the mixed beta-1,3; beta-1,6-polysaccharides such as carboxymethyl-pachyman, yeast glucan and laminarin . Both cellulase I and the beta-glucosidase degraded xylan, and it is proposed that the xylanase activity is an inherent feature of these two enzymes . Lichenin (beta-1,4; beta-1,3) was degraded by all three cellulases . Cellulase II cannot degrade carboxymethyl-cellulose, and with filter paper as substrate the end product was cellobiose, which indicates that cellulase II is an exo-beta-1,4-glucan cellobiosylhydrolase . Degradation of cellulose (filter paper) can be catalysed independently by each of the three cellulases; there was no synergistic effect between any of the cellulases, and cellobiose was the principal product of degradation . The mode of action of one cellulase (cellulase III) was examined by using reduced cellulodextrins . The central linkages of the cellulodextrins were the preferred points of cleavage, which, with the rapid decrease in viscosity of carboxymethyl-cellulose, confirmed that cellulase III was an endocellulase . The rate of hydrolysis increased with chain length of the reduced cellulodextrins, and these kinetic data indicated that the specificity region of cellulase III was five or six glucose units in length.

J Gen Virol, 1981 Jan, 52(Pt 1), 141 - 4
Properties of the virulent form of a mitomycin C- or temperature-induced thermophilic bacteriophage; Holmes D et al.; A virulent bacteriophage was isolated from lysates of Bacillus stearothermophilus strain NU-10 induced either by treatment with mitomycin C or by shifts in temperature . Optimum conditions for induction, morphology and other properties of the virus are described . Chloroform treatment and shifts in temperature of producer cells released approximately similar amounts of phage as did mitomycin induction, suggesting an effect on release rather than on synthesis of the virus.

Nucleic Acids Res, 1980 Dec 11, 8(23), 5551 - 66
In vitro splicing of the ribosomal RNA precursor in isolated nucleoli from Tetrahymena; Carin M et al.; The macronuclear rRNA genes of Tetrahymena thermophila contain an 0.4 kb intervening sequence in the 26S rRNA coding region . The sequence is represented within the primary transcription product . We demonstrate in this paper that the enzyme activities necessary for the endonucleolytic cleavage as well as for the ligation of the transcript are associated with the isolated . The intervening sequence is excised as an unique molecule, which is stable in vitro . About 50% of the in vitro synthesized RNA is processed . Faithful in vitro transcription occurs in the presence of the divalent ions Mg2+, Mn2+ and Co2+ while processing takes place only in the presence of Mg2+ . The absolute requirement for Mg2+ in the excision reaction enables us to synthesize labelled pre-rRNA in the presence of Mn2+ or Co2+ . The synthesized RNA can be used as a substrate in studies of th processing enzymes in vitro.

Nucleic Acids Res, 1980 Dec 11, 8(23), 5611 - 22
Nuclease TT1 from Thermus thermophilus HB8 has an endonuclease activity preferential to circular DNAs; Takahashi M et al.; Homogeneously purified nuclease TT1 from Thermus thermophilus HB8 is known as an exonuclease to produce 5'-mononucleotides . Besides the exonuclease activity, nuclease TT1 also possesses endonuclease activity preferential to superhelical (form I) and single-stranded circular DNA . Although the rate of cleavage is slower than that of form I, covalently closed circular DNA (form I') is also cleaved . Form I DNA was nicked to yield relaxed circles (form II) first, and was then nicked at the opposite site to yield unit length linear DNA (form III) which was subsequently hydrolyzed to 5'-mononucleotides exonucleolytically . Both endo- and exo-nuclease activities co-migrate on polyacrylamide gels . The general properties of the endonuclease activity are very similar to those of the exonuclease activity . The temperature optimum for endonuclease activity was 85 degrees C . The pH-optimum was in pH-range from 7.5-9.1 . The enzyme was active over a wide range of Mg2+ concentrations (2.5-125 mM), and was inhibited by EDTA . A linear substrate such as (dT)8 was a competitive inhibitor for this endonuclease activity.

J Biochem (Tokyo), 1980 Dec, 88(6), 1895 - 8
Thermostability and aliphatic index of globular proteins; Ikai A; A statistical analysis shows that the aliphatic index, which is defined as the relative volume of a protein occupied by aliphatic side chains (alanine, valine, isoleucine, and leucine), of proteins of thermophilic bacteria is significantly higher than that of ordinary proteins . The index may be regarded as a positive factor for the increase of thermostability of globular proteins.

Eur J Biochem, 1980 Dec, 113(1), 159 - 73
Purification from Tetrahymena thermophila of DNA polymerase and a protein which modifies its activity; Ganz PR et al.; Two proteins, which may be involved in DNA replication, have been isolated and characterized from the eukaryote Tetrahymena thermophila . One of these proteins, DNA polymerase, has been purified to apparent homogeneity . The enzyme has a native molecular weight of approximately 90 000 in the presence of salt and aggregates to higher-molecular-weight forms in the absence of salt . Purified preparations of the enzyme yield a major subunit of Mr 45 000 when the protein is analyzed by denaturing electrophoresis . Tetrahymena DNA polymerase requires a divalent cation for catalysis and prefers gapped template-primers over denatured and native DNAs . A template-primer such as poly(dT) . oligo(A) can also be elongated by the DNA polymerase . However, the enzyme will not use poly(A) . oligo(dT) as a template-primer . Sulfhydryl-blocking reagents, such as N-ethylmaleimide, inhibit Tetrahymena DNA polymerase . The DNA polymerase lacks assayable levels of both single and double-stranded deoxyribonuclease activity . Throughout the early stages of purification the DNA polymerase chromatographs together with a protein of molecular weight 100 000 . This protein, which yields a single major polypeptide of Mr 25 000 when analyzed by denaturing electrophoresis, has single-stranded-DNA-binding properties and has the ability to stimulate both the rate and extent of DNA-polymerase-catalyzed DNA synthesis in vitro . By virtue of this latter ability, the protein has been referred to as the M (for 'modifying') protein . Maximum stimulation of DNA polymerase was achieved with template-primers, which contained large stretches of single-stranded template such as poly(dA) . (dT)10 mixed in a template-to-primer ratio of one to one . Stimulation of DNA polymerase activity by M protein in vitro appears to involve formation of longer product DNA.

J Gen Microbiol, 1980 Dec, 121(Pt . 2), 311 - 7
Temperature characteristics and Arrhenius plots for nominal psychrophiles, mesophiles and thermophiles; Mohr PW et al.; The specific growth rates at various temperatures of 12 bacterial species were measured and plotted as Arrhenius profiles . Temperature characteristics and optimum temperatures for maximum specific growth rates were estimated from these curves . The data reveal that one of two forms of the Arrhenius profile is characteristic of each bacterium: one curve is a simple smooth curve with a single predominant slope at sub-optimum temperatures; the other is a more complex curve with two distinct slopes at sub-optimum temperatures . The simple curves describes bacteria across the entire biokinetic range whereas the more complex curve occurs only with organisms which have optimum temperatures for replication above 37 degrees C . Describing bacteria in terms of these forms of the Arrhenius profile is less arbitrary than is categorization based on optimum temperatures.

Biochim Biophys Acta, 1980 Nov 5, 593(1), 11 - 6
Functional arginine residues and carboxyl groups in the adenosine triphosphatase of the thermophilic bacterium PS-3; Arana JL et al.; Treatment of purified ATPase of the thermophilic bacterium PS-3 with the arginine reagent phenylglyoxal or with Woodward's reagent K, gave complete inactivation of the enzyme . The inactivation rates followed apparent first-order kinetics . The apparent order of reaction with respect to inhibitor concentrations gave values near to 1 with both reagents, suggesting that inactivation was a consequence of modifying one arginine or carboxyl group per active site . ADP and ATP strongly protected the thermophilic ATPase against both reagents . GDP and IDP protected less, whilst CTP did not protect . Experiments in which the incorporation of {14C}phenylglyoxal into the enzyme was measured show that extrapolation of incorporation to 100% inactivation of the enzyme gives 8-9 mol {14C}phenylglyoxal per mol ATPase, whilst ADP or ATP prevent modification of about one arginine per mol.

Ann Microbiol (Paris), 1980 Nov-Dec, 131B(3), 289 - 96
Solubilization of insoluble phosphates by thermophilic fungi; Singh CP et al.; The solubilization of tricalcium phosphate and rock phosphate and assimilation of solubilized P by thermophilic fungi isolated from compost were studied . The solubilization of tricalcium phosphate was greater than that of rock phosphate on inoculation with fungi in liquid medium, but growth of most of the fungi was greater in rock phosphate . Torula thermophila solubilized tricalcium phosphate maximally . There was solubilization of rock phosphate in semi-solid lignocellulose medium by Aspergillus fumigatus.

J Dairy Sci, 1980 Nov, 63(11), 1953 - 6
Methane production from cattle waste in laboratory reactors at 40 degrees and 60 degrees C after solid-liquid separation; Rorick MB et al.; Whole dairy waste and liquid effluent separated from the same waste with a solid-liquid separator were fermented at mesophilic and thermophilic temperatures . Chemical analyses of the two materials were similar . Methane production was superior in thermophilic reactors . With subtrates adjusted to 4.1% volatile solids, average methane production at 60 degrees C (166 ml/g volatile solids fed to reactors at 3- and 6-day retention time) was as efficient as the 40 degrees C (162 ml/g at 5- and 10-day retention times) . Thermophilic reactors produced 1.67 liter methane/liter reactor per day as compared to .93 liter for mesophilic reactors . Efficiency of methanogenesis was no greater for whole waste than for separated effluent . Production of methane for the two substrates averaged over retention times and temperatures was 156 ml/g volatile solids fed to reactor for whole waste and 173 ml/g for separated effluent.

Biochem J, 1980 Nov 1, 191(2), 457 - 65
Purification of an NADH-(dichlorophenol-indophenol) oxidoreductase from Bacillus stearothermophilus; Mains I et al.; An NADH-(dichlorophenol-indophenol) oxidoreductase was purified 104-fold and in 25% overall yield from the thermophilic bacterium Bacillus stearothermophilus, strain PH24 . After solubilization in 2M-NaCl at 70 degrees C, the enzyme was purified by ion-exchange and hydroxyapatite chromatography, followed by affinity chromatography on immobilized Cibacron Blue 3GA . The purified enzyme had a mol.wt . of 43 000 and had an absorption spectrum characteristic of flavoprotein . The enzyme activity was enhanced by FMN and by CN- . The enzyme was inhibited by EDTA and by rho-chloromercuribenzoic acid.

J Biol Chem, 1980 Oct 10, 255(19), 9507 - 16
Two glutamine synthetases from Bacillus caldolyticus, an extreme thermophile . Isolation, physicochemical and kinetic properties; Wedler FC et al.; Two distinctly different glutamine synthetase enzymes (EI and EII) have been isolated from the extreme thermophile Bacillus caldolyticus, grown on chemically defined medium at 70 degrees C . Purification to homogeneity mainly involves affinity chromatography and heat treatment with substrate protection . Biosynthesis of total enzyme activity can be repressed by at least 8-fold by high ammonia, with synthesis of EI being repressed more strongly than EII . A variety of chemical and biochemical tests failed to provide evidence for regulation of EI or EII by covalent modification, e.g . proteolysis, phosphorylation, or adenylylation . Neither of the thermophiic enzymes will cross-react with antibodies for the Escherichia coli or Bacillus subtilis glutamine synthetases . Both enzymes are composed of 12 subunits, each approximately 51,000 daltons . However, EI and EII differ significantly in their amino acid composition, isoelectric points (5.2 and 5.5, respectively), rates of migration on polyacrylamide electrophoresis gels at pH 6.8, and kinetic properties, EI is more active with Mg(II) than with Mn(II), but EII is more active with Mn(II) than Mg(II) . Cd(II) activates EII more than EI, and only EI shows activity with Co(II) . For both enzymes, the Mn(II)-stimulated activity is optimal at pH 6.0 to 6.5, with Mn(II)/ATP = 1.0, but the pH optimum with Mg(II) is near pH 7.5, however, with a ratio of Mg(II)/ATP > 2 . Substrate Km values at 70 degrees C differ for EI versus EII but are quite comparable to those seen for mesophilic glutamine synthetases . Studies with structural analogs of substrates indicate that active site specificity is maintained at extreme temperatures: substitution of alpha-OH for alpha-HN2 is allowed, but unfavorable changes occur upon substitution of methyl groups for the alpha-H or onto the alpha-NH2 of L-Glu, and for D-Glu or L-Asp . EII is almost absdolutely specific for ATP, but EI can also use ITP, GTP, and UTP as substrates to some extent . The divalent metal ion that is present can affect both specificity for analogs and substrate Km values . Kinetic binding plots (v versus {S}) are biphasic for NH3 and L-Glu with the more active forms of each enzyme, EI-Mg and EII-Mn, respectively; but no positive cooperativity is observed . ATP binding is strictly hyperbolic, in contrast to the positive cooperativity previously observed with other Bacillus sp . enzymes . For purified EI and EII, Arrhenius plots are nonlinear with Mn(II) or Mg(II), exhibiting slope changes in the range of 55-65 degrees C; however, for EI-EII mixtures in crude cell extracts these plots are nearly linear.

Biochem J, 1980 Oct 1, 191(1), 83 - 94
Purification and properties of the cellulases from the thermophilic fungus Thermoascus aurantiacus; Tong CC et al.; Three cellulases and a beta-glucosidase were purified from the culture filtrate of the thermophilic fungus Thermoascus aurantiacus . The isolated enzymes were all homogeneous on polyacrylamide-disc-gel electrophoresis . Data from chromatography on Bio-Gel P-60 and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis indicated mol.wts . of 87000 (beta-glucosidase), 78000 (cellulase I), 49000 (cellulase II) and 34000 (cellulase III); the carbohydrate contents of the enzymes were 33.0, 5.5, 2.6 and 1.8% (w/w) respectively . Although the three purified cellulases were active towards filter paper, only cellulases I and III were active towards CM(carboxymethyl)-cellulose . Cellulase I was also active towards yeast glucan . The Km and catalytic-centre-activity values for the enzymes were as follows; 0.52 mumol/ml and 6.5 X 10(4) for beta-glucosidase on p-nitrophenyl beta-D-glucoside, 3.9 mg/ml and 6.3 for cellulase I on CM-cellulose, 1.2 mg/ml and 1.1 for cellulase I on yeast glucan, 35.5 mg/ml and 0.34 for cellulase II on filter paper, and 1.9 mg/ml and 33 for cellulase III on CM-cellulose.

Biochim Biophys Acta, 1980 Sep 9, 615(1), 34 - 47
L-alanine dehydrogenase from Thermus thermophilus; Vali Z et al.; A heat-stable L-alanine dehydrogenase was isolated and purified from the extremely thermophilic microorganism, Thermus thermophilus, by affinity chromatography . The enzyme has a molecular weight of 290 000, as determined by the sedimentation equilibrium method, and is composed of six subunits of identical molecular weight as concluded from sodium dodecyl sulphate gel electrophoresis . The enzyme has been characterized in terms of pH- and substrate concentration-dependence of activity, substrate specificity, inhibition by D-alanine and D-cysteine and amino acid composition . The parameters obtained are very similar to those reported for L-alanine dehydrogenase from the mesophilic microorganism, Bacillus subtilis (Yoshida, A . and Freese, E . (1965) Biochim . Biophys . Acta 96, 248--262) . The thermal stability of the T . thermophilus enzyme is much greater than that of the B . subtilis enzyme . Activation free energy (delta G), activation enthalpy (delta H) and activation entropy (delta S) values were determined for both the alanine deamination and for the heat inactivation reactions of the thermophilic and mesophilic enzymes . The values obtained for the catalytic reaction were practically equal . However, the two enzymes differed significantly in these parameters determined for the enzyme inactivation, which indicates that the factors ensuring the thermoresistance of the enzyme from T . thermophilus do not affect enzyme activity.

Mikrobiologiia, 1980 Sep-Oct, 49(5), 715 - 21
{Possibility of DNA-ligase participation in regulating the synthesis and degradation of heat-damaged DNA in permeable Bacillus stearothermophilus cells}; Trofimenko AF et al.; The chromosome of Bacillus stearothermophilus was found to contain apurine regions which were repaired mainly by excisions . In the permeable cells of the thermophilic bacterium, the observed intensive degradation and resynthesis of DNA were regulated by active DNA ligase . In this case, NAD (the cofactor of ligase) sharply inhibited the ATP-dependent and independent synthesis of DNA . Apparently, DNA ligase may be involved in the regulation of degradation and resynthesis of DNA with thermal lesions in the cells of thermophilic organisms.

Am Rev Respir Dis, 1980 Sep, 122(3), 437 - 43
Serologically detectable HLA-A, B, and C loci antigens in farmer's lung disease; Flaherty DK et al.; The frequency of serologically detectable (SD) HLA-A, B, and C loci antigens in subjects with farmer's lung disease (N = 100) was compared with that of age- and sex-matched normal normal farmers with no precipitating antibodies to extracts of thermophilic actinomycetes . A subset of the farming population with antibodies to the thermophilic actinomycetes and no evidence of clinical disease (N = 55) was also agae- and sex-matched to the farmer's lung disease population . No significant associations between any of the SD HLA antigens tested and farmer's lung disease were found in the study . The data demonstrated that there was no association between SD HLA antigens and farmer's lung disease in random populations.

J Biochem (Tokyo), 1980 Sep, 88(3), 737 - 47
A DNA methylase from Thermus thermophilus HB8; Sato S et al.; A DNA methylase was purified in a homogeneous state from a extremely thermophilic bacterium, Thermus thermophilus HB8, by chromatography on, successively, phosphocellulose, CM-cellulose, and heparin-Sepharose . The molecular weight of the enzyme was determined to be about 44,000 by gel filtration on a Sephadex G-100 column and 41,000 by SDS-poly-acrylamide gel electrophoresis, and these findings suggest a single polypeptide enzyme . The enzyme develops maximum activity around pH 7.4 and at 70 degrees C . Enzymatic activity is completely inhibited by 0.2 M NaCl or 2 mM HgCl2 . The enzyme transfers methyl groups from S-adenosyl-L-methionine to a double stranded DNA . The sole product of the reaction was identified as N-6-methyl adenine after hydrolysis of the DNA with formic acid . The enzyme kinetics obey the Michaelis-Menten equation and Km values for S-adenosylmethionine and lambda phage DNA were determined to be 0.8 muM and 10 microgram/ml, respectively . The enzyme does not transfer methyl groups to TthHB8I endonuclease digested DNA as well as the host (T . thermophilus HB8) DNA . The number of methyl groups of the fully methylated phiX174 RF DNA was about twice as many as TthHB8I endonuclease sites on the DNA . The distribution of the methyl groups of phiX174 RF DNA among the HaeIII fragments was the same as that of TthHB8I endonuclease sites, suggesting that this DNA methylase is the other component of the modification-restriction system including TthHB8I endonuclease . The enzyme probably recognizes the sequence, 5'-TCGA-3', in a double stranded DNA and probably methylates adenine in the above sequence.

Eur J Biochem, 1980 Sep, 110(1), 217 - 23
Proton nuclear-magnetic-resonance and resonance Raman studies of thermophilic cytochrome c-552 from Thermus thermophilus HB8; Hon-Nami K et al.; The pH and temperature dependences of the 270-MHz proton nuclear magnetic resonance and resonance Raman spectra of Thermus thermophilus cytochrome c-552 were studied . Observation of the NMR methyl signal of the iron-bound methionine indicates that a methionine residue is the sixth ligand of heme iron in both ferric and ferrous states, although the environment of this methionine is not similar to that in mitochondrial cytochrome c . The NMR methyl signal of the coordinated methionine in the ferrous state was observed even at 87 degrees C, indicating the retention of the methionine ligand at the sixth coordination position . None of resonance Raman lines in ferrous cytochrome c-552 at higher temperatures showed a prominant temperature-dependent frequency shift, which implies that the heme iron was still bound with strong ligands and retained the low-spin state . In either redox state overall thermal denaturation did not occur even at 87 degrees C, although the ferric form existed in thermal spin mixture of the low-spin and high-spin species at higher temperatures . The hyperfine-shifted NMR resonances of the ferric form indicated rapid exchange of the sixth ligand at alkaline pH in the process of a single-step alkaline isomerization.

J Biochem (Tokyo), 1980 Sep, 88(3), 715 - 24
A study of the interaction between promotor DNA and T . thermophilus DNA-dependent RNA polymerase 1,2; Tsuji S et al.; As the first step in the process of RNA synthesis, RNA polymerase binds to a specific site (promoter) and forms an open complex . In this process, it is considered that the structure of DNA is changed to an unidentified form . We investigated the structure of this DNA, which consists of an open complex, by means of CD . For this purpose, we used very stable RNA polymerase (of T . thermophilus HB8) and the fd-RF-DNA fragment (Hap-Hga V), which has only one promoter . We have confirmed that the complex of the holo enzyme with Hap-Hga V fragment at 50 degrees C is an open complex . We obtained the CD spectral difference between the open complex and its constituents for the first time . The observed CD difference spectra in the UV region (250-300 nm) were compared with the theoretical difference CD . It was deduced that the DNA of the open complex may be melted around the initiation point over a rather longer range than expected.

Biochim Biophys Acta, 1980 Aug 26, 609(1), 180 - 95
Nucleoprotein subunit structure in an unusual prokaryotic organism: Thermoplasma acidophilum; Searcy DG et al.; The freeliving thermophilic mycoplasma, Thermoplasma acidophilum, has a small acid-soluble protein tightly bound to its DNA . This protein is similar to eukaryotic histones in both size and amino acid composition . Here we report that the protein condenses DNA into globular particles that are about half the size of eukaryotic nucleosomes . Our conclusions are based primarily upon the following observations: (1) Nuclease digestion produced DNA fragments of 40 base-pairs . (2) The ratio of protein to DNA was such that 4--5 molecules of protein were associated with each 40 base-pair segment of DNA . (3) Protein crosslinking reagents produced tetramers of the histone-like protein . (4) Electron microscopy revealed globular particles 5--6 nm in diameter . (5) Each globular particle reduced the apparent contour length of the DNA by 40 base-pairs . Thus, each nucleoprotein particle is apparently composed of 40 base-pairs of DNA coiled around four molecules of proteins.

Biochem J, 1980 Aug 15, 190(2), 457 - 60
Phosphate utilization and constitutive synthesis of phosphatases in Thermoactinomyces vulgaris Tsilinsky; Sinha U et al.; Thermoactinomyces vulgaris utilized both organic and inorganic phosphates with equal efficiency for its growth . The specific activities of the thermophilic acid and alkaline phosphatases were found to be maximum at 1 mM concentration of each phosphate source . All the phosphatase isoenzymes (three alkaline and one acidic) were observed irrespective of the substrate source and concentration, suggesting constitutive synthesis of the enzymes . During growth and differentiation, both acid and alkaline phosphatases exhibited uniformly stable patterns of isoenzymes with slight variations in their specific activities.

Nucleic Acids Res, 1980 Aug 11, 8(15), 3275 - 85
A second site specific endonuclease from Thermus thermophilus 111, Tth111II; Shinomiya T et al.; A second site specific endonuclease with novel specificity has been purified from Thermus thermophilus strain 111 and named Tth111II . The enzyme is active at temperature up to 80 degrees C and requires Mg2+ or Mn2+ for endonuclease activity . Tth111II cleaves phi X174RFDNA into 11 fragments and lambda NA into more than 25 fragments . From the 5'-terminal sequences of TthlllII fragments of phi X174RFDNA determined by the two dimensional homochromatography and the survey on nucleotide sequence of phi X174RFDNA, it was concluded that Tth111II recognizes the DNA sequence (see former index) and cleaves the sites as indicated by the arrows.

J Cell Sci, 1980 Aug, 44, 317 - 33
The oral apparatus of Tetrahymena . V . Oral apparatus polypeptides and their distribution; Gavin RH; Two-dimensional electrophoresis was used to resolve approximately 162 polypeptides from the isolated oral apparatus of Tetrahymena thermophila . The molecular weight range was between 110 000 and 15 000 Daltons . The polypeptides had apparent isoelectric points between pH 3.3 and pH 7.2 . Electrophoretic analysis of isolated ciliary axonemes and fractionated oral apparatuses made possible the assignment of polypeptides to structures within the oral apparatus . Approximately 24 polypeptides, including alpha and beta tubulins, are probable components of the basal body-basal plate complex . At least 5 of the oral apparatus polypeptides, including alpha and beta tubulin, are components of the oral apparatus ciliary axonemes . Approximately 138 polypeptides are components of the oral apparatus framework.

Acta Pathol Microbiol Scand {B}, 1980 Aug, 88(4), 207 - 18
Serology of Campylobacter fetus ss . jejuni )"related" campylobacters) . Demonstration of strain-specific and interstrain-related antigens by immunoelectrophoresis and co-agglutination; Kosunen TU et al.; Rabbit antisera against two strains of Campylobacter fetus ss . fetus (serotype A), two strains of C . fetus ss . intestinalis (serotypes A and B respectively), and eight stains of C . fetus ss . jejuni were used in serological studies of these strains with the use of co-agglutination (COA), line-rocket immunoelectrophoresis (L-RIE) and rocket-line immunoelectrophoresis (R-LIE) . Whole bacterial cells, either heated at 56 degrees C . boiled or atuoclaved, were used in COA tests . Unheated sonicates were used in L-RIE, and sonicates, unheated, boiled or autoclaved, in R-LIE . The antigenic properties of C . fetus ss . fetus and C . fetus ss . intestinalis were distinctly different from those of the thermophilic C . fetus ss . jejuni strains as shown both by COA and L-RIE . Serotypes A and B of the two former species were also differentiated . In COA tests the C . fetus ss . jejui=ni organisms gave the strongest reactions with homologous antibodies, but several interstrain cross-reactions were seen . By absorption strain specific COA reagents were obtained . Several reactions of identity indicating cross-reactive antigens were also seen with L-RIE within the ss . jejuni group . These results generally agreed with those obtained by COA . With the use of unheated, boiled or autoclaved organisms or sonicates heat labile antigens were differentiated from heat stable ones with the use of COA and R-LIE . The apparent antigenic heterogeneity of Campylobacteria indicates the importance of their serological grouping, e.g . for clinical and epidemiological investigations . COA and immunoelectrophoresis techniques can be effectively applied for such studies.

J Bioenerg Biomembr, 1980 Aug, 12(3-4), 297 - 308
A stable Na+/H+ antiporter of thermophilic bacterium PS3; Goto K et al.; As a first step in the isolation of a stable Na+/H+ antiporter, its reaction in sonicated membrane vesicles of thermophilic bacterium PS3 has been characterized . The sonicated vesicles showed quenching of quinacrine fluorescence in either NADH oxidation or ATP hydrolysis . The quenching was reversed by the addition of Na+, Li+, Mn2+, Cd2+, and Co2+, but not of choline+ or Ca2+, regardless of their counter anions . 22Na+ was taken up into the vesicles by NADH oxidation, and the 22Na+ uptake was inhibited by the addition of an uncoupler . H+ release was observed on addition of Na+ to sonicated vesicles . The magnitude of the pH difference across the membrane induced by NADH oxidation was constant at pH 7.0 to 9.1, but the Na+/H+ antiport was affected by the pH of the medium (optimum pH-8.5) . The Km's of the antiporter for Na+ and Li+ were 2.5 and 0.1 mM, respectively, but the Vmax values for the two ions were the same at pH 8.0 . In the presence of Li+, no further decrease of fluorescence quenching was observed on addition of Na+ and vice versa . The Na+/H+ antiporter activity in PS3 was stable at 70 degrees C, and the optimum temperature for activity was 55-60 degrees C . In contrast to mesophilic cation/H+ antiporters, this antiporter was not inhibited by a thiol reagent.

J Bacteriol, 1980 Aug, 143(2), 693 - 702
Biochemical correlations among the thermophilic enteric yeasts Torulopsis bovina, Torulopsis pintolopesii, Saccharomyces telluris, and Candida slooffii; Watson K et al.; Spontaneous and drug-induced respiration-deficient mutants were isolated from the thermophilic enteric yeasts Torulopsis bovina and Saccharomyces telluris . The biochemical properties of these yeasts were compared with those of the two naturally occurring respiration-deficient thermophilic yeasts T . pintolopesii and Candida slooffii . Succinate dehydrogenase was not detected in mitochondrial fractions from C . slooffii, but was present in all other species . Cytochrome c oxidase, succinate oxidase, and reduced nicotinamide adenine dinucleotide oxidase were not detected in C . slooffii, T . pintolopesii, and the respiration-deficient mutants . Low-temperature cytochrome spectra revealed the presence of cytochromes aa3, b, c1, and c in T . bovina and S . telluris; cytochromes b, c1, and c in C . slooffii and T . pintolopesii; and cytochromes c1 and c in the spontaneous respiration-deficient mutants . Palmitoleic and oleic acids were the major fatty acids in all the species . It was noteworthy that T . pintolopesii was rich in lauric and myristic acids . CsCl equilibrium centrifugation experiments showed the presence in all the yeasts of a light-buoyant-density (1.6785 to 1.6837-g/cm3) deoxyribonucleic acid band which was identified as mitochondrial deoxyribonucleic acid by its selective elimination on treatment of cells with ethidium bromide . The latter result indicated that the spontaneous respiration-deficient mutants were similar to cytoplasmic petite mutants of S . cerevisiae . Although classical assimilation and fermentation tests indicated that the spontaneous respiration-deficient mutants were strains of T . pintolopesii, it was concluded, on the basis of marked physiological and biochemical differences, that this was not the case.

J Protozool, 1980 Aug, 27(3), 253 - 7
Morphologic and biochemical characterization of Crithidia brasiliensis sp . n; De Sa MF et al.; A trypanosomatid with a choanomastigote stage and, therefore, belonging to the genus Crithidia, was isolated in culture from the alimentary tract of the hemipteran genus Zelus . The trypanosomatid was able to grow at 37 C, a characteristic reported to date from only 2 other members of Crithidia, C hutneri and C . luciliae thermophila . Subsequently, the flagellate was cloned for biochemical studies which involved cleaving of kDNA by restriction endonucleases and analyses of the isoenzyme and histone patterns . In all the attributes revealed by the foregoing methods, the organism from Zelus differed from the latter 2 congeneric species . On these and morphologic grounds, this organism appears to belong in a new species for which the name Crithidia brasiliensis sp . n . is proposed.

J Protozool, 1980 Aug, 27(3), 339 - 41
Production of melanin precursors by a mutant of Tetrahymena thermophila; Kaney AR et al.; A mutant of Tetrahymena thermophila, "pig," excretes melanin precursors into the culture medium where spontaneous polymerization to melanin occurs . The precursors, probably oxidation products of catecholamines, are produced in large amounts by the mutant by decarboxylation of tyrosine and hydroxylation of the resulting tyramine . Overproduction and excretion of precursors by the mutant appears to result from elevated specific activity of L-aromatic amino acid decarboxylase (DOPA decarboxylase) (E.C . 4.1.1.26).

Biochim Biophys Acta, 1980 Jul 14, 619(1), 68 - 75
Phospholipid composition and heat sensitivity in a thermophilic bacterium; Merkel GJ et al.; A Gram-negative thermophilic bacterium designated strain LEH-1, survived under non-growth conditions, temperatures 5 degrees C above its maximum for growth when acetate or n-heptadecane served as carbon source, whereas cells grown with glucose or glycerol were markedly more sensitive to elevated temperatures . The total extractable lipids from strain LEH-1 and the concentrations of phosphatidylethanolamine and phosphatidic acid in the cytoplasmic membranes was altered by the growth substrate . Growth with acetate of n-heptadecane as carbon source yielded cells containing more phosphatidic acid than phosphatidylethanolamine . Conversely, more phosphatidylethanolamine was present in cells following growth with glucose or glycerol as carbon source . The relative amount of specific fatty acids and distribution in major phospholipid moieties was also affected by the growth substrate . These differences in composition may reflect an alteration in the physical properties of the cellular membrane and account for the divergence in heat sensitivity.

Clin Allergy, 1980 Jul, 10(4), 405 - 11
Circulating immune complexes in bronchial asthma; Stendardi L et al.; Soluble immune complexes were detected by a sold phase Clq binding assay in forty-two out of 106 well studied asthmatic patients (39.6%) and in eleven out of 145 age-matched controls (8%; P < 0.01) . Clinical significance of immune complexes has been evaluated by comparing the following parameters in patients with and without such complexes: age, sex, duration of disease, IgE-mediated allergy (RAST, skin test), precipitins (Aspergillus, Candida albicans, Thermoactinomycetes), corticodependency, lung function tests, associated symptoms (hivernal bronchitis, urticaria, eczema,...), desenstization treatment, serum concentration in immunoglobulin in G, A, M and E . Immune complexes were found more frequently in female than in male patients . The prevalence of immune complexes was higher in patients treated by hyposensitization therapy and in cases with precipitating antibodies against thermophilic actinomycetes and Aspergillus.

Cell, 1980 Jul, 20(3), 609 - 17
Histone variants specific to the transcriptionally active, amitotically dividing macronucleus of the unicellular eucaryote, Tetrahymena thermophila; Allis CD et al.; Two dimensional gel electrophoresis (triton-acid-urea followed by SDS) has been used to resolve two previously uncharacterized, quantitatively minor histone variants in acid extracts from macronuclei of Tetrahymena thermophila . Utilizing techniques which allow characterization of these variants without purifying them in significant quantities, we identify one protein as a subtype of H3 . The other protein is a moderately lysine-rich histone whose tryptic peptide map differs from that of both H2A and H2B . However, its pattern of secondary modifications, its detergent-binding properties and its methionineless nature all suggest that it is more like H2A than any other histone . Both variants are associated with nucleosomes derived from macronuclei . Thus primary sequence variants of the inner histones, presumably indicative of nucleosome heterogeneity, exist in a lower eucaryote, in an amitotic nucleus, and within the nucleus of a clonally propagated organism . Evidence is presented that these newly described minor variants are absent in micronuclei, suggesting that they play an important role in the structural and functional differentiation of macronuclear chromatin.

Eur J Biochem, 1980 Jul, 108(2), 581 - 6
Heat stability of a tetrameric enzyme, D-glyceraldehyde-3-phosphate dehydrogenase; Walker JE et al.; The tetrameric enzyme D-glyceraldehyde-3-phosphate dehydrogenase from the moderate thermophile Bacillus stearothermophilus is more stable to thermal denaturation than its counterpart from lobster muscle {Harris et al . (1980) Eur . J . Biochem . 108, 535-547} . Extra buried ionic bonds between subunits of the thermophilic enzyme make an important contribution to thermal stabilisation . Further stabilisatio of the tetrameric enzyme is derived from additional hydrophobic interactions between the S-loops at the core of the tetramer . In the enzyme from the extreme thermophile Thermus aquaticus, which is even more thermostable, intersubunit ion pairs must also play a role but changes in interactions at the surface appear to be equally important . Thus additional hydrophobic interactions at the edge of subunit interfaces would prevent access of water to the interior of the molecule . Furthermore, the arrangement of charged residues on the surface of the T . aquaticus enzyme would allow maximal surface ion pair formation . The presence of surface ion pairs in other proteins correlates well with thermal stability {Perutz, M . F . and Raidt, H . (1975) Nature (Lond.) 255, 256-258} and would provide a general stabilising influence on the subunit in this case.

Eur J Biochem, 1980 Jul, 108(2), 549 - 65
D-glyceraldehyde-3-phosphate dehydrogenase . Complete amino-acid sequence of the enzyme from Bacillus stearothermophilus; Walker JE et al.; 1 . The complete amino acid sequence of D-glyceraldehyde-3-phosphate dehydrogenase from the moderate thermophile Bacillus stearothermophilus has been determined . 2 . This has been achieved largely by the automated sequence analysis of large fragements derived by chemical cleavage with cyanogen bromide, BNPS-skatole {the product of reaction between N-bromosuccinimide and 2-(nitrophenyl-sulphenyl)-3-methylindole} and hydroxylamine and enzymic hydrolysis with trypsin at arginine residues . 3 . The sequence is as follows: (See Text) . It has been numbered to maximise homology with the four complete sequences of this enzyme from other sources . Hence the N-terminal residue is numberd 0 and two deletions and two insertions have been introduced . 4 . The inability of the B . stearothermophilus apo-enzyme to transfer an acyl moiety from Cys-149 to Lys-183 oberved with muscle enzymes is explained by the replacement of lysine by arginine in the enzyme from the thermophilic organism . 5 . The sequences of the S-loop regions, which form the core of the tetrameric enzyme, are similar to each other in B . stearothermophilus and Thermus aquaticus and differ from the highly conserved S-loops of three enzymes from mesophiles.

Eur J Biochem, 1980 Jul, 108(2), 535 - 47
D-glyceraldehyde-3-phosphate dehydrogenase . The purification and characterisation of the enzyme from the thermophiles Bacillus stearothermophilus and Thermus aquaticus; Harris JI et al.; 1 . D-Glyceraldehyde-3-phosphate dehydrogenase from two thermophilic bacteria has been purified by procedures including affinity chromatography on NAD+-Sepharose . 2 . Methods for making NAD+-free enzyme are also described . 3 . Both the holo and apo forms of the enzyme from Bacillus stearothermophilus have been crystallised . 4 . The enzymes are tetrameric and composed of four chemically identical polypeptide chains of molecular weight 36,000 . 5 . The enzymes are much more stable to heat than their counterparts from mesophiles.

Gene, 1980 Jul, 10(2), 137 - 45
Cloning and expression of the leucine gene from Thermus thermophilus in Escherichia coli; Nagahari K et al.; A pBR322-T . leu hybrid plasmid was constructed which contains a 3.75 Md HindIII-fragment derived from Thermus thermophilus HB27 chromosomal DNA . In the Escherichia coli host, this plasmid coded for the beta-IPM dehydrogenase (product of leuB) activity, the optimal temperature of which was 80 degrees C, suggesting that information on the thermostability of the enzyme lies in its structural gene . 10-day propagation of E . coli {pBR322-T.leu} at 37 degrees C decreased the temperature optimum from 80 degrees to 75 degrees C . This change, which was found to depend on the plasmid but not on the host cells, might be due to selection of some mutation at the non-restrictive temperature of 37 degrees C . Our results suggest that the 3.75 Md HindIII-fragment of pBR322-T.leu carries a promoter of the thermophile, which could function in E . coli.

Eur J Biochem, 1980 Jul, 108(2), 567 - 79
D-glyceraldehyde-3-phosphate dehydrogenase . Amino-acid sequence of the enzyme from the extreme thermophile Thermus aquaticus; Hocking JD et al.; 1 . The amino acid sequence of D-glyceraldehyde-3-phosphate dehydrogenase from the extreme thermophile Thermus aquaticus has been elucidated . 2 . The polypeptide contains 332 amino acids and its sequence is 70% identical with that of the enzyme from the moderate thermophile Bacillus stearothermophilus . 3 . In contrast to less thermostable forms of the enzymes from B . stearothermophilus, pig, lobster and yeast, the T . aquaticus enzyme has only one cysteine residue, namely cysteine-149 which is required for catalysis.

J Biochem (Tokyo), 1980 Jul, 88(1), 59 - 68
Substrate specificity of nuclease TT1 from Thermus thermophilus HB8; Takahashi M; The substrate and the action mechanism of a nuclease named nuclease TT1, from the culture broth of an extreme thermophile, Thermus thermophilus HB8, were investigated . The enzyme is nonspecific for the sugar moiety and cleaves both single- and double-stranded DNAs, rRNA, tRNA and oligonucleotides irrespective of chain length to produce 5'-mononucleotides exonucleolitically . The action mechanism is processive and the enzyme shows no porality of degradation . The minimal unit as a substrate is a 5'-dinucleotide . The rate of hydrolysis is independent of a terminal phosphate group . The substrate lacking a 5'-phosphoryl group is degraded to leave the 5'-terminus and the penultimate nucleotide (NpN) as a core . The substrate possessing a 3'-phosphoryl group is degraded to leave the mononucleoside 5',3'-diphosphates (pNp) . However, NpN and pNp are gradually degraded by a large dose of the enzyme to produce a 5'-mononucleotide . The enzyme is free from nonspecific phosphatase and phosphodiesterase activities . Application of this enzyme to determine the sequence of oligonucleotides is shown.

Eur J Biochem, 1980 Jul, 108(2), 599 - 611
Primary structure of triosephosphate isomerase from Bacillus stearothermophilus; Artavanis-Tsakonas S et al.; 1 . Triosephosphate isomerase from Bacillus stearothermophilus is a dimeric enzyme comprising two chemically identical polypeptide chains . 2 . The nearly complete amino acid sequence of the subunit polypeptide chain has been established from sequences of tryptic, chymotryptic and lysine-blocked tyrptic fragments of S-{2-14C}carboxymethylated enzyme . Overlaps not established by experimental data have been provisionally established from considerations of sequence homology with previously established sequences for the rabbit, chicken and coelacanth enzymes . The nearly complete sequence of the 249 residues is as follows . (See Text) . 3 . Comparison of the thermophile and chicken muscle enzymes shows that 40% of the residues are in identical sequence . 4 . Correlation of the sequence of the thermophile enzyme with the three-dimensional structure of the muscle enzyme shows that residues in the catalytic site and in the subunit interface are strongly conserved . Possible correlations between sequence changes and thermal stabilisation of the dimeric structure are also noted.

J Membr Biol, 1980 Jun 30, 55(1), 1 - 8
Energy-transducing proteins in thermophilic biomembranes; Kagawa Y; Biomembranes are the major site of energy transduction . The chemisomotic theroy of energy transduction is based on the following four major systems (i) H+-ATPase which is composed of a catalytic portion (F1) and a H+-channel (Fo), (ii) electron transport components, (iii) H+-linked porters, and (iv) a H+-impermeable lipid bilayer which is plugged through by systems i to iii that are specially oriented to translocate H+ . Studies on the molecular mechanism of energy transduction have been hampered by the impurity, instability and complexity of preparations of membrane proteins from mesophilic organism . However, using stable, simple membrane proteins from a thermophilic bacterium, we obtained the following results: 1) Thermophilic H+-ATPase was dissociated into 5 subunits of F1 and 3 subunits of Fo and their functions and structures were studied by reconstitution . F1 was crystallized . 2) Thermophilic cytochrome oxidase, cytochrome c and NADH-dehydrogenase were purified . In contrast to the complex mitochondrial cytochrome oxidase (7 subunits) and NADH-dehydrogenase (3 subunits), the purified thermophilic proteins were shown to be composed of single components . 3) H+-linked porters such as a H+-driven amino acid carrier and a Na+-H+ antiporter were characterized . 4) Thermophilic lipids were shown to be completely saturated . Using these stable lipids, liposomes capable of H+-driven vectorial reactions including net ATP synthesis and alanine transport were reconstituted.

Biochemistry, 1980 Jun 24, 19(13), 2873 - 82
Superoxide dismutase from Bacillus stearothermophilus . Complete amino acid sequence of a manganese enzyme; Brock CJ et al.; Superoxide dismutase from the thermophilic bacterium Bacillus stearothermophilus is dimeric with a molecular weight of 45 487 . It contains one atom manganese(III) per dimeric molecule {Brock, C . J., Harris, J . I., & Sato, S . (1976) J . Mol . Biol . 107, 175-178} . The subunits are identical, and the following primary structure comprising 203 amino acids has been determined: (Formula: see text) The enzyme is identical in 60% of its residues with the Escherichia coli B manganese enzyme {Steinman, H . M . (1978) J . Biol . Chem 253, 8708-8720} . Neither manganese enzyme has significant homology with the Cu/Zn superoxide dismutase from bovine erythrocytes . The secondary structures of the manganese enzymes predicted by McLachlan's method {McLachlan, A . D . (1977) Int.J . Quantum chem . 12, 371-385} indicate that the eight-stranded beta barrel of the Cu/Zn enzyme {Richardson, J.S., Thomas, K . A., Byron, H . R., & Richardson, D . C . (1975) Proc . Natl . Acad . Sci . U.S.A . 72, 1349-1353} is absent from the manganese enzymes.

Biochem J, 1980 Jun 15, 188(3), 695 - 704
Regeneration of cilia in starved Tetrahymena thermophila involves induced synthesis of ciliary proteins but not synthesis of membrane lipids; Skriver L et al.; The synthesis of ciliary-membrane phospholipids and ciliary proteins was studied after deciliation in starving Tetrahymena thermophila cells . Deciliated cells regenerated the new ciliary membrane without any induced phospholipid synthesis . The constant cell volume found during the regrowth of the cilia suggests that renewal of ciliary membranes takes place by insertion of intracellular membrane material into the cell surface . In contrast with the absence of induced phospholipid synthesis during ciliary regeneration, the synthesis of ciliary proteins was found to be induced . This enhanced synthetic activity was made possible by an increased rate of intracellular protein degradation in regenerating cells . It was found that the extent of the induced synthesis strongly depends upon the growth conditions of the cells before starvation . Furthermore, it was shown that the degree of induced protein synthesis is greater for higher-molecular-weight ciliary proteins than for lower-molecular-weight species.

Biochim Biophys Acta, 1980 Jun 13, 613(2), 249 - 55
Formation and energy transfer of a fluorescent derivative of B . stearothermophilus glyceraldehyde-3-phosphate dehydrogenase; Ho YS et al.; The active site carboxymethylated glyceraldehyde-3-phosphate dehydrogenase from B . stearothermophilus when irradiated with ultraviolet light in the presence of NAD gives rise to a fluorescent derivative closely similar to that obtained from the muscle enzyme in fluorescence properties . A radiationless energy transfer also occurs between the tryptophan residues of the enzyme protein and the new fluorophore, as for the muscle enzyme . Quantitative determinations of the quantum yields and calculations according to the Forster equation five a distance of 26.36 A between the tryptophan residues and the new fluorophore . In contrast to the muscle enzyme, the irradiated thermophilic enzyme contains four fluorescent NAD derivatives per enzyme tetramer as shown by phosphorus analysis.

J Cell Sci, 1980 Jun, 43, 75 - 91
The relationship between the excess-delay phenomenon and temperature-sensitive periods in Tetrahymena thermophila; Frankel J et al.; Although temperatures of 37.5 and 39 degrees C allow continuous and rapid exponential growth of wild type Tetrahymena thermophila, sudden shifts up to these temperatures can bring about long excess-delays of cell division with accompanying resorption of developing oral primordia . A characteristic parameter of this delay-phenomenon is the physiological transition point, before which delays are maximal and after which they are negligible . When measured at a restrictive temperature that does not induce excess delays (36 degrees C), the end of the temperature-sensitive period of the cell division arrest of mutant cdaA1 precedes the physiological transition point, that of cdaH1 roughly coincides with it, while the entire temperature-sensitive period of cdaC2 comes after the physiological transition point . When cdaA1 cells are exposed to 37.5 degrees C or above, the manifestations of temperature sensitivity are drastically affected: the estimate of the end of the temperature-sensitive period (the execution point) becomes spuriously late, and the characteristic division arrest following heat shocks is not manifested . The differential effects of the higher restrictive temperatures on cdaH1 are most subtle, whereas those on cdaC2 are negligible . We conclude that the excess-delay phenomenon involves a set-back of genemediated processes occurring at specific stages of the cell cycle.

J Cell Sci, 1980 Jun, 43, 59 - 74
Temperature-sensitive periods of mutations affecting cell division in Tetrahymena thermophila; Frankel J et al.; Temperature-sensitive periods were determined by application of temperature shifts and shocks to 3 temperature-sensitive cell division arrest (cda) mutants of Tetrahymena thermophila . A restrictive temperature, 36 degrees C, was found at which all 3 mutants are fully penetrant, yet other physiological effects are minimal . At this temperature, the temperature-sensitive period of cdaC2 is a unique 5-min period in mid-division, that of cdaA1 is a similarly brief period situated about 0.5 h prior to cell division, while the temperature-sensitive period of cdaH1 is 20 to 30 min long and immediately precedes cell division . These periods either coincide with (cdaC2, cdaH1) or immediately precede (cdaA1) the onset of phenotypic abnormality at the restrictive temperature . Brief exposure to 36 degrees C during the temperature-sensitive period in any of these mutants brings about irreversible arrest of division furrows in progress or preparation . Mutant cells suffering such arrest can, however, divide again at a permissive temperature by forming new furrows at different sites.

Orig Life, 1980 Jun, 10(2), 185 - 92
Purification and properties of malate dehydrogenase from the extreme thermophile Bacillus caldolyticus; Kristjansson H et al.; The enzyme malate dehydrogenase (EC 1.1.1.37) from an extreme thermophile B . Caldolyticus was purified to about 91% homogeneity . The molar mass of the enzyme was determined as 73 000 daltons and it is composed of two subunits, each with a molar mass of 37 000 . Initial velocity studies with oxaloacetic acid and NADH as substrates at pH 8.1, over a range of temperatures, indicates that the enzyme operates via a sequential type mechanism . Van't Hoff plots of the kinetic parameters displayed sharp changes in slope at characteristic temperatures, whereas the Arrhenius plot exhibited no such breaks over the temperature interval investigated . The enzyme was found to be stable at 41 degrees C and lower temperatures . At 51 degrees C and 59 degrees C an almost immediate 20% reduction in activity was obtained, but no further inactivation occurred during the 60 min of incubation . At 59 degrees C the enzyme lost 50% of its initial activity in about 38 s . High concentration of NADH was observed to greatly stabilize the enzyme at that temperature . It is suggested that the slope changes in the Van't Hoff plots and the stability profiles at 51 degrees C and 59 degrees C are representative of a temperature induced conformational change in the enzyme.

J Biochem (Tokyo), 1980 Jun, 87(6), 1609 - 17
Nucleotide binding to isolated alpha and beta subunits of proton translocating adenosine triphosphatase studied with circular dichroism; Ohta S et al.; The catalytic and allosteric sites of proton translocating adenosine triphosphatase (ATPase) were studied by measuring the binding of nucleotides to the ATPase, and its alpha and beta subunits purified from thermophilic bacterium PS3, with a circular dichroic spectrometer . In contrast to mesophilic ATPases, this thermophilic enzmye contained no tightly bound nucleotides, and its subunits were stable after their purification . These properties were advantageous for analyzing both catalytic and allosteric sites . The former site showed rapid and loose binding, but the latter slow (t 1/2 = 1 h, for ADP) and tight binding . When a nucleotide was bound, the beta subunits showed a negative ellipticity at 275 nm corresponding to a tyrosyl residue, while the alpha subunits showed an ellipticity change corresponding to the absorption curve of the bound nucleotide . This difference enabled us to distinguish the binding sites in ATPase . At a low concentration, ADP selectively bound to alpha subunits in the ATPase, while at a high concentration, it bound to both subunits . This finding suggests that the tight binding sites are located in the alpha subunits . Although ADP and ATP bound to both the purified alpha and beta subunits, CTP did not bind to beta but only to alpha subunits, and ITP bound to beta but hardly to alpha . These nucleotide specificities also supported the idea that the catalytic sites are located in the beta subunits and the allosteric sites are located in the alpha subunits.

J Biol Chem, 1980 May 25, 255(10), 4691 - 7
The effect of amino acid starvation on nucleoside uptake and RNA synthesis in Tetrahymena; Campbell JM et al.; The uptake of nucleosides and the synthesis of RNA in Tetrahymena thermophila were examined following amino acid starvation . Omission of leucine, phenylalanine, or arginine from the medium resulted in a rapid decrease in the incorporation of {3H}uridine into the acid-soluble pool and acid-insoluble material (RNA) . Amino acid starvation inhibited the uptake of all ribo- and deoxyribonucleosides tested but did not affect the uptake of amino acids or glucose . In addition, under the conditions used, the omission of an amino acid did not result in a large decrease in amino acid incorporation into total protein . Treatment of cells with cycloheximide or emetine gave results similar to the effects of amino acid starvation, but in these experiments the inhibition of protein synthesis was essentially complete . Nucleotide pool sizes were also measured following amino acid starvation . ATP and UTP levels were essentially unchanged, but the dTTP pool size was decreased by 40% . The decrease in RNA synthesis in vivo in the absence of an essential amino acid was reflected in the endogenous RNA synthetic activity of isolated nuclei . However, when solubilized RNA polymerase activity was measured with calf thymus DNA as template, no significant difference was observed between control and amino acid-starved cells.

Biochem J, 1980 May 15, 188(2), 351 - 61
Photosynthetic electron transport in a cell-free preparation from the thermophilic blue-green alga Phormidium laminosum; Stewart AC et al.; 1 . A cell-free preparation of membrane fragments was prepared from the thermophilic blue-green alga Phormidium laminosum by lysozyme treatment of the cells followed by osmotic shock to lyse the spheroplasts . The membrane fragments showed high rates of photosynthetic electron transport and O2 evolution (180-250 mumol of O2/h per mg of chlorophyll a with 2,6-dimethyl-1,4-benzoquinone as electron acceptor) . O2-evolution activity was stable provided that cations (e.g . 10mM-Mg2+ or 100mM-Na+) or glycerol (25%, v/v) were present in the suspending medium . 2 . The components of the electron-transport chain in P . laminosum were similar to those of other blue-green algae: the cells contained Pigment P700, plastocyanin, soluble high-potential cytochrome c-553, soluble low-potential cytochrome c-54 and membrane-bound cytochromes f, b-563 and b-559 (both low- and high-potential forms) . The amounts and midpoint potentials of the membrane-bound cytochromes were similar to those in higher-plant chloroplasts . 3 . Although O2 evolution in P . laminosum spheroplasts was resistant to high temperatures, thermal stability was not retained in the cell-free preparation . However, in contrast with higher plants, O2 evolution in P . laminosum membrane fragments was remarkably resistant to the non-ionic detergent Triton X-100.

Biochemistry, 1980 May 13, 19(10), 2017 - 22
Malate dehydrogenase from thermophilic and mesophilic bacteria . Molecular size, subunit structure, amino acid composition, immunochemical homology, and catalytic activity; Sundaram TK et al.; Malate dehydrogenases isolated from a number of mesophilic, moderately thermophilic, and extremely thermophilic bacteria yield upon denaturation subunits of molecular weight 32 000--36 000 . Determination of their native molecular weights shows that some of the enzymes are dimeric and others are tetrameric; the two types are distributed in each of the three classes of bacteria . The amino acid compositions of the enzymes show no consistent trend that can be related to the progression of thermostability from the mesophile through the moderate thermophile to the extreme thermophile species . The tetrameric enzyme species all exhibit a high level of structural homology as judged by the criterion of immunological cross-reaction . Little cross-reaction occurs, however, between the tetramers and the dimers . The dimeric enzyme from the extreme thermophile, Thermus aquaticus, cross-reacts only weakly, if at all, even with dimeric malate dehydrogenases . The catalytic activities of the malate dehydrogenases vary over a wide range . Potassium chloride, organic solvents such as acetone, and the protein denaturants urea and guanidine hydrochloride activate a number of the malate dehydrogenases under the assay conditions employed . The diversity among the bacterial malate dehydrogenases, manifested not only in molecular size and subunit structure but also in properties such as catalytic activity and the dependence of this activity on electrolytes, organic solvents, and denaturants, indicates significant structural differences between several of these cognate enzyme species.

Biochemistry, 1980 May 13, 19(10), 2209 - 15
Proton nuclear magnetic resonance study on the roles of histidine residues in the binding of polypeptide chain elongation factor Tu from Thermus thermophilus with aminoacyl transfer ribonucleic acid and guanine nucleotides; Nakano A et al.; Proton nuclear magnetic resonance (1H NMR) spectra were measured of the polypeptide chain elongation factor Tu (EF-Tu) from an extreme thermophile, Thermus thermophilus HB8 {Nakano, A., Miyazawa, T., Nakamura, S., & Kaziro, Y . (1979) Arch . Biochem . Biophys . 196, 233-238}, in order to elucidate the environment around functionally important histidine residues . In the present study, the behavior of five histidine C2 proton signals was studied in more detail . A hydrogen-deuterium exchange experiment was carried out on the histidine C2 protons of free EF-Tu, and the previous assignments of C2 proton signals were revised in part . An analysis of the 1H NMR spectra of EF-Tu photooxidized under various conditions indicates that a histidine residue is located in the aminoacyl-tRNA binding site and is probably essential for the binding with aminoacyl-tRNA . A solvent-accessible histidine residue is found to lie near the aminoacyl-tRNA binding site . Furthermore, the effect of paramagnetic hexacyanochromate(III) ion on the 1H NMR spectra of free EF-Tu suggests that another histidine residue lies near the guanine nucleotide binding site.

Biochemistry, 1980 May 13, 19(10), 2160 - 5
Intersubunit interactions in proton-translocating adenosine triphosphatase as revealed by hydrogen-exchange kinetics; Ohta S et al.; The rates of hydrogen-deuterium exchange in the peptide groups of the alpha and beta subunits and the alpha-beta subunit complex of proton-translocating adenosine triphosphatase from the thermophilic bacterium PS3 were examined . The exchange was found to be much slower in the isolated beta subunit than in the isolated alpha subunit . This has been taken as indicating that the structure of the beta subunit is tighter than that of the alpha subunit . Adenosine 5'-triphosphate (ATP) caused tightening of a relatively tight portion of the alpha subunit and of a relatively loose portion of the beta subunit . When the alpha and beta subunits are brought into contact, tightening of the alpha subunit, but not the beta subunit, occurs . The effect of ATP on the structure of the beta subunit is more pronounced in the presence of the alpha subunit than in its absence . These findings support the idea proposed previously that the alpha subunit has an allosteric site and the beta subunit a catalytic site and that the conformation of the beta subunit is controlled by the alpha subunit.

Prikl Biokhim Mikrobiol, 1980 May-Jun, 16(3), 356 - 62
{Effect of products of thermophilic methane fermentation on the conidial activation and citric acid biosynthesis by the fungus Aspergillus niger}; Mushnikova LN et al.; The effect of products of thermophilic methane fermentation (PTMF) and their hydrolyzates on the conidial germination and citric acid bioysnthesis by the fungus Aspergillus niger when cultivated on the synthetic and molasses media was investigated . An addition of the above products increased the amount of germinated conidia on the synthetic medium by 1.2--2 times and on the molasses medium by 1.1--1.4 times . The citric acid yeild per unit surface area on the two media grew by 60--100% and 10--20%, respectively . Optimal concentration of dry preparations of PTMF was 5--7.5 mg/100 ml and that of their hydrolyzates was 0.5--1.5 ml/100 ml of the medium . An addition of PTMF to the molasses medium resulted both in an increased yeild of citric acid (1500--1700 g/m2/day) and in reduced (by 12--24 hours) time of the biosynthesis process.

Mikrobiologiia, 1980 May-Jun, 49(3), 493 - 6
{Functional characteristics of the membrane preparations from the cells of the thermophilic hydrogen bacterium . Pseudomonas thermophila}; Romanova AK et al.; The electron transport chain (ETC) of Pseudomonas thermophila K-2 was examined by the amperometric determination of O2 uptake by the preparations of membranes isolated by ultracentrifugation at 14,000 g . Amytal and cyanide were found to inhibit endogenous respiration of membranes from freshly grown cells . The membrane preparations, after exhaustion of endogenous substrates in them, oxidized NADH and succinate at rates of 4.00 and 0.83 mumole/min per 1 mg of membrane protein, respectively . The oxidation of NADH was inhibited by rotenone and cyanide, while the oxidation of succinate, only by cyanide . Maxima corresponding to NADH and iron-containing proteins were found in the fluorescence spectra of membrane preparations from Ps . thermophila K-2 . Endogenous NADH was not susceptible either to incubation of the preparations in the air, or to hydrogen being bubbled through . These results and the data reported in literature make it possible to conclude that the membrane preparations from Ps . thermophila K-2 contain all the ETC components similar to the mitochondrial ones.

Mycopathologia, 1980 May 1, 71(1), 9 - 16
Thermophilic mycoflora of cigarettes and cured tobacco leaves; Ogundero VW; Nine species of thermophilic fungi were obtained from retailed packets of imported and locally manufactured brands of cigarettes and from cured tobacco leaves . They include known human pathogens such as Thermoascus aurantiacus Miehe sensu Apinis, Mucor pursillus Lindt and Mucor miehei Cooney and Emerson . Chaetomium thermophile La Touche, Humicola insolens Cooney and Emerson, Thermoascus crustaeus (Apinis and Chesters) Stolk and Mucor miehei have not been previously reported on tobacco products . Fewer species were obtained from foreign cigarettes than from locally manufactured brands . A mean moisture content of 19% was recorded for imported cigarettes and 37% for Nigerian brands . The potential health hazards posed to man by the unrestricted use of tobacco products are discussed.

Cell, 1980 May, 20(1), 55 - 64
Proteolytic processing of histone H3 in chromatin: a physiologically regulated event in Tetrahymena micronuclei; Allis CD et al.; Micronuclei of Tetrahymena thermophila contain two electrophoretically distinct forms of histone H3 . The slower migrating micronuclear species, H3S, is indistinguishable from the macronuclear H3 by electrophoretic analyses in three gel systems and by partial proteolytic peptide mapping . The faster species, H3F, is unique to micronuclei . Pulse-chase experiments with radioactive amino acids show that H3S is a precursor to H3F . We present evidence that the in vivo processing of H3S into H3F requires cell growth and/or division and may occur regularly each generation at a specific point in the cell cycle . The processing event must occur after H3F is deposited on micronuclear chromatin, since both H3S and H3F can be isolated from sucrose gradient-purified mononucleosomes (Allis, Glover and Gorovsky, 1979) . Partial proteolytic peptide mapping coupled with 3H-N-ethylmaleimide labeling suggest that the processing event involves a proteolytic cleavage from the amino terminal end of H3F . Automated sequence analyses of 14C-lysine-labeled macronuclear H3 together with either 3H-lysine-labeled H3S or H3F demonstrated that H3F is derived from H3S by a proteolytic cleavage which removes six residues from the amino terminus . These observations represent the first demonstration of a physiologically regulated proteolytic processing event in histone metabolism.

J Cell Sci, 1980 Apr, 42, 247 - 60
Mitochondrial associations with specific microtubular components of the cortex of Tetrahymena thermophila . II . Response of the mitochondrial pattern to changes in the microtubule pattern; Aufderheide KJ; Mitochondria in Tetrahymena thermophila are known to associate with the cell cortex in asymmetrical patterns corresponding to the asymmetrical organization of the microtubular components of the cortex . Specific mitochondrion-microtubule associations are seen at the light-microscope and ultrastructural levels . The hypothesis that the mitochondrial pattern is determined by the microtubular pattern was tested . Using the phenotypes of various mutations to generate changes in the organization of the cortex, the response of the mitochondrial pattern to the cortical change was assayed . The results consistently show that changes in the local organization of the cortical microtubule systems are followed by a corresponding change in the cortical mitochondrial pattern . However, the mitochondrial pattern is unaffected by a mutation which changes the 'long-distance' patterning of certain organelles, but which does not influence the local microtubular organization . Thus, the data support the hypothesis that there is a causal relationship between the arrangement of the cortical microtubules and the corresponding mitochondrial pattern.

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