Thermophile

J Protozool, 1987 Nov, 34(4), 435 - 8
Effect of biogenic amines on phagocytosis in Tetrahymena thermophila; Quinones-Maldonado V et al.; Stimulation of phagocytosis by serotonin and catecholamines in Tetrahymena grown in proteose-peptone medium proved to be concentration dependent, the optimal concentrations being approximately 0.1 to 1.0 microM . The serotonergic antagonists, spiperone, and metergoline, also stimulated the process, whereas the beta- and alpha-adrenergic antagonists, propranolol, alprenolol, and ergocryptine, had no effect or inhibited phagocytosis . A wide variety of derivatives of the biogenic amines had no effect on phagocytosis, demonstrating the specificity of recognition mechanism for neurohormones in Tetrahymena . Such hormones act by at least two independent mechanisms, one for adrenergic agonists, another for dopamine . Presumably, recognition mechanisms for hormones in protozoa resemble in some respects those in multicellular organisms, therefore bespeaking a common origin.

J Exp Biol, 1987 Nov, 133, 169 - 82
Thermal dependence of passive electrical properties of lizard muscle fibres; Adams BA; 1 . The thermal dependence of passive electrical properties was determined for twitch fibres from the white region of the iliofibularis (IF) muscle of Anolis cristatellus (15-35 degrees C) and Sceloporus occidentalis (15-40 degrees C), and for twitch fibres from the white (15-45 degrees C) and red (15-40 degrees C) regions of the IF of Dipsosaurus dorsalis . These species differ in thermal ecology, with Anolis being the least thermophilic and Dipsosaurus the most thermophilic . 2 . Iliofibularis fibres from the three species reacted similarly to changing temperature . As temperature was increased, input resistance (Rin) decreased (average R10 = 0.7), length constant (L) decreased (average R10 = 0.9), time constant (tau) decreased (average R10 = 0.8), sarcoplasmic resistivity (Rs) decreased (average R10 = 0.8) and apparent membrane resistance (Rm) decreased (average R10 = 0.7) . In contrast, apparent membrane capacitance (Cm) increased with increasing temperature (average R10 = 1.3) . 3 . Rin, L, tau and apparent Rm were lowest in fibres from Anolis (the least thermophilic species) and highest in fibres from Dipsosaurus (the most thermophilic species) . Anolis had the largest and Dipsosaurus the smallest diameter fibres (126 and 57 micron, respectively) . Apparent Cm was highest in fibres from Sceloporus, which had fibres of intermediate diameter (101 micron) . Rs did not differ significantly among species . 4 . The effect of temperature on the passive electrical properties of these lizard fibres was similar to that reported for muscle fibres from other ectothermic animals (crustaceans, insects, fish and amphibians) but qualitatively different from that reported for some mammalian (cat tenuissimus, goat intercostal) fibres . The changes that occur in the passive electrical properties render the fibres less excitable as temperature increases.

J Protozool, 1987 Nov, 34(4), 416 - 7
Self-splicing RNA and an RNA enzyme in Tetrahymena; Zaug AJ et al.; The RNA molecules transcribed from many eukaryotic genes are interrupted by intervening sequences, which are removed by a process called RNA splicing . One structurally related group of intervening sequences, the group I intervening sequences, are found in a variety of microorganisms . Some of these, including the group I intervening sequence from the ribosomal RNA precursor of Tetrahymena thermophila, have been shown to mediate their own splicing in an RNA-catalyzed reaction . Following its excision from the ribosomal RNA precursor, the Tetrahymena intervening sequence acts as an enzyme, cutting and rejoining RNA substrates.

Biochem Int, 1987 Nov, 15(5), 953 - 60
Three-dimensional crystals of ribosomes and their subunits from eu- and archaebacteria; Glotz C et al.; Ordered three-dimensional crystals of 70S ribosomes as well as of 30S and 50S ribosomal subunits from various bacteria (E . coli, Bacillus stearothermophilus, Thermus thermophilus and Halobacterium marismortui) have been grown by vapour diffusion in hanging drops using mono- and polyalcohols . A new compact crystal form of 50S subunits has been obtained, and it is suitable for crystallographic studies at medium resolution . In addition, from one crystal form large crystals could be grown in X-ray capillaries . In all cases the crystals were obtained from functionally active ribosomal particles, and the particles from dissolved crystals retained their integrity and biological activity.

Genetics, 1987 Nov, 117(3), 451 - 66
Genomic organization and developmental fate of adjacent repeated sequences in a foldback DNA clone of Tetrahymena thermophila; Tschunko AH et al.; DNA sequence elimination and rearrangement occurs during the development of somatic cell lineages of eukaryotes and was first discovered over a century ago . However, the significance and mechanism of chromatin elimination are not understood . DNA elimination also occurs during the development of the somatic macronucleus from the germinal micronucleus in unicellular ciliated protozoa such as Tetrahymena thermophila . In this study foldback DNA from the micronucleus was used as a probe to isolate ten clones . All of those tested (4/4) contained sequences that were repetitive in the micronucleus and rearranged in the macronucleus . The presence of inverted repeated sequences was clearly demonstrated in one of them by electron microscopy . DNA sequence analysis showed that the left portion of this clone contains three tandem, directly repeated copies of a 340-bp sequence, a 120-bp portion of which appears in inverted orientation at a 1.6-kb distance . This clone, pTtFB1, was subjected to a detailed analysis of its developmental fate . Subregions were subcloned and used as probes against Southern blots of micronuclear and macronuclear DNA . We found that all subregions defined repeated sequence families in the micronuclear genome . A minimum of four different families was defined, two of which are retained in the macronucleus and two of which are completely eliminated . The inverted repeat family is retained with little rearrangement . Two of the families, defined by subregions that do not contain parts of the inverted repeat, one in the "loop" and one in the "right flanking region," are totally eliminated during macronuclear development--and contain open reading frames . A fourth family occurs in the "loop" region and is rearranged extensively during development . The two gene families that are eliminated are stable in the micronuclear genome but are not clustered together as evidenced by experiments in which DNAs from nullisomic strains are used to map family members to specific micronuclear chromosomes . The inverted repeat family is also stable in the micronuclear genome and is dispersed among several chromosomes . The significance of retained inverted repeats to the process of elimination is discussed.

J Biol Chem, 1987 Oct 25, 262(30), 14672 - 82
Site-directed mutagenesis of core sequence elements 9R', 9L, 9R, and 2 in self-splicing Tetrahymena pre-rRNA; Williamson CL et al.; The intron within the Tetrahymena thermophila nuclear large rRNA precursor is the best studied example of group I self-splicing introns . In this paper, we examine the structural and functional roles of four internal sequence elements which are characteristic of group I introns in the RNA-catalyzed processing reactions . Oligonucleotide-directed mutagenesis was used to generate mutations in sequence elements 9R', 9L, 9R and 2 of the Tetrahymena intervening sequence . Self-splicing activities of variant precursor RNAs were characterized by in vitro splicing following transcription with T7 or SP6 RNA polymerase . First, we confirm the proposed base pairing of sequence elements 9R and 9R' by construction and analysis of compensatory mutations . Mutations in elements 9R (G272A C274G) and 9R' (G100C C102U) each disrupt the pairing and eliminate self-splicing activity . A compensatory 9R/9R' mutation (G100C C102U G272A C274G) restores pairing and normal splicing activity . We conclude that 9R X 9R' pairing is a requirement for self-splicing . Second, we show that self-splicing activity is very sensitive to both nucleotide sequence and RNA secondary structure in the pairing segments of elements 9L and 2 . Mutations within these regions at positions 266, 268, 307, and 309 can increase as well as decrease activity relative to wild type . Third, a mutation in the highly conserved nonpairing segment of element 9L (U259A A261C) increases KM for GTP from 29 to 120 microM, but does not otherwise affect splicing activity . The primary consequence of this mutation is a decrease in GTP binding energy of approximately 0.9 kcal/mol . Last, we show that a mutation in the highly conserved nonpairing segment of element 2 (A301C A302G G303C) eliminates transesterification activity, but does not affect 3' splice site hydrolysis.

FEBS Lett, 1987 Oct 19, 223(1), 92 - 6
Amino acid sequence of iron-superoxide dismutase from Pseudomonas ovalis; Isobe T et al.; The amino acid sequence of iron-superoxide dismutase from Pseudomonas ovalis was deduced by the analyses of peptides derived from limited hydrolysis of the aminoethylated or pyridylethylated apoprotein with trypsin, Staphylococcus aureus V8 protease, and dilute acid hydrolysis . The polypeptide chain contains 195 amino acid residues and has a calculated Mr of 21,421 . The sequence is highly homologous (65% identity) to the recently published sequence of the iron-superoxide dismutase from Photobacterium leiognathi . It is also homologous to the known sequences of the manganese-superoxide dismutase by sharing 33-53% identical residues . Alignment of the superoxide dismutase sequences and the available structural information from X-ray crystallography suggest that the ligands to the iron in the P . ovalis superoxide dismutase are His-26, His-74, Asp-156 and His-160, which align with the ligands to the manganese in the Thermus thermophilus manganese-superoxide dismutase . The sequence information of the P . ovalis dismutase will facilitate refinement of the X-ray crystallographic data that are now available at 2.9 A resolution.

Biochem Biophys Res Commun, 1987 Oct 14, 148(1), 397 - 402
A 31P-NMR study on multilamellar liposomes formed from the lipids of a thermophilic bacterium; Pinheiro TJ et al.; The membrane lipids of a thermophilic bacterium, Thermus SPS11, isolated from thermal springs in Sao Pedro do Sul, Portugal, were fractionated by chromatography on silica gel . The total lipid extract was found to contain one major phospholipid (PL), which accounts for about 90% of the total lipid phosphorous, and one major glycolipid (GL), which accounts for about 95% of the total carbohydrate in the non-phospholipid fraction . The membranes also contain about 11% by weight of a complex mixture of carotenoids (CA) . Multilamellar liposomes, in excess water, formed from PL and mixtures of PL with GL and CA in proportions found in the natural membrane were investigated by proton-decoupled 31P-nuclear magnetic resonance (NMR) spectroscopy and X-ray diffraction . All mixtures examined were found to be in a lamellar phase with disordered hydrophobic chains with no evidence for "non-bilayer structures" between 23 degrees and 85 degrees C . Compared to bilayers formed from pure PL or mixtures of PL and CA, significantly larger values for the chemical shift anisotropy of the 31P-NMR powder patterns were obtained from bilayers formed from mixtures of PL and GL, at temperatures above 75 degrees C, and mixtures of PL, GL and CA at all temperatures examined . These differences are interpreted in terms of changes in the order of the bilayer and/or changes in the orientation of the phosphate moiety of PL . The significance of these results to the thermophily of the bacterium is discussed.

Nucleic Acids Res, 1987 Oct 12, 15(19), 7735 - 47
Sequences implicated in the processing of Thermus thermophilus HB8 23S rRNA; Hartmann RK et al.; Nuclease S1 mapping analyses were performed in order to detect processing intermediates of pre-23S rRNA from Thermus thermophilus HB8 . Two processing sites were identified downstream the start of transcription and several consecutive cleavage sites are associated with the mature 5'-end . In the 3'-flanking region one "primary" site and two cleavages which generate short-living intermediates were detected . A series of successive intermediates in the region of the mature 3'-end implies the existence of--in analogy to Escherichia coli--a 3'-exonucleolytic activity . The data were correlated with potential secondary structures within the pre-23S rRNA, which exhibit various repeated sequence elements . M13 sequencing data support the existence of one secondary structural element associated with the strong "primary" cleavage site in the 3'-flanking region . In T . thermophilus we can exclude the formation of an extended base-paired and precursor-specific stem enclosing the 23S rRNA which is inferred to mediate recognition by RNase III in E . coli.

Epidemiol Infect, 1987 Oct, 99(2), 275 - 82
Serogroups of thermophilic campylobacters from humans and from non-human sources, Israel 1982-1985; Rogol M et al.; The distribution of serogroups of thermophilic campylobacters isolated in Israel from human patients (2421 isolates), chicken (942), turkeys (158), cattle (398), wild birds (234) and other sources, was studied . Among the human isolates, 74 ROG-serogroups were identified . The six most commonly isolated of these (1, 18; 11; 12; 8,23; 4 and 5,39) were found frequently in chickens . Only four common serogroups in man were also common in cattle, three in turkeys and two in wild birds . Two common serogroups in man (1,18 and 5,39) were prevalent all over the country, while others were regionally distributed . When the prevalence of different serogroups in Israel was compared to that in Canada, some groups were common to both countries and others were common in only one or the other . Campylobacter jejuni accounted for 86.7% to 92.1% of the isolates from man, chickens, turkeys, cattle and most of the wild birds . C . coli was found in 34.4% of isolates from cattle egrets and in 76.5% of those from pigs.

Epidemiol Infect, 1987 Oct, 99(2), 265 - 74
Survival of thermophilic campylobacters on fingertips and their elimination by washing and disinfection; Coates D et al.; A simple impression-plate technique has been used to investigate the survival of four thermophilic campylobacter strains applied to fingertips . Campylobacters suspended in 0.1% peptone water and dried on the fingertips survived for different periods of time ranging from less than 1 to greater than or equal to 4 min . However, campylobacters suspended in chicken liquor or blood survived for much longer periods . The most resilient organism was Campylobacter jejuni NCTC 11392 which, when suspended in 50% horse blood, survived for an hour . Suspensions containing 10(6)-10(7) organisms prepared in 50% blood and dried on to fingertips were removed by thorough hand washing with either soap and water or water alone followed by drying on paper towels, but persisted on wet hands . The organisms were also eliminated by wiping the hands with a tissue saturated with 70% isopropyl alcohol for 15 sec.

Appl Environ Microbiol, 1987 Oct, 53(10), 2414 - 9
Difference in sporogenous bacterial populations in thermophilic (55 degrees C) and mesophilic (35 degrees C) anaerobic sewage digestion; Chen M; Spores, sporeforming vegetative cells, and asporogenous populations were enumerated in two semicontinuous anaerobic fermentors digesting municipal primary sludge at 35 and 55 degrees C for more than 87 days . In the 35 degrees C fermentor, the anaerobic total population was 312.5 X 10(6)/ml, with 25.0 X 10(6)/ml being sporogenous . The populations that digest casein, starch, pectin, and cellulose were 23.1 X 10(6), 59.2 X 10(6), 26.2 X 10(6), and 7.3 X 10(6)/ml, respectively, with 2.8 X 10(6), 6.7 X 10(6), 3.4 X 10(6), and 1.5 X 10(6)/ml being sporogenous, respectively . The sporeformers accounted for 8.0 to 20.0% of each of the respective populations . In the 55 degrees C fermentor, the anaerobic total population was 512.5 X 10(6)/ml, with 336.6 X 10(6)/ml being sporogenous . The populations that digest casein, starch, pectin, and cellulose were 97.7 X 10(6), 190.7 X 10(6), 75.8 X 10(6), and 11.2 X 10(6)/ml, respectively, with 47.8 X 10(6), 110.6 X 10(6), 43.3 X 10(6), and 5.1 X 10(6)/ml, respectively, being sporogenous . The sporeformers represented 45.5 to 65.7% of each of the respective populations . The numbers of thermophilic sporeforming vegetative cells in the 55 degrees C fermentor were 9.0 to 19.8 times higher than their counterparts in the 35 degrees C fermentor . Most sporeformers were in the vegetative state in the 35 and 55 degrees C fermentors . After 18 days of fermentation at 55 degrees C, sporeformers carried out most of the digestion; however, the digestion was shared by both sporeformers and asporogenous bacteria after 87 days of fermentation . In the 35 degrees C fermentor, asporogenous bacteria digested most of the sludge . During the 18- and 87-day experimental periods, sporeformers were never predominant.

Bioorg Khim, 1987 Oct, 13(10), 1344 - 50
{Interaction of s4U8 region of tRNA Phe with tRNA-(adenine-1-)-methyltransferase from Thermus thermophilus}; Venkstern TV et al.; Photoaffinity labelling of tRNA (adenine-1-)-methyltransferase with an E . coli tRNA(Phe) derivative bearing 4-azidophenylmercuro group attached to s4U residue as well as direct photocross-linking of the native tRNA(Phe) with the enzyme via s4U residue has been studied . Both techniques labelling gave similar results, leading to covalent attachment of tRNA(Phe) to the enzyme within a specific complex . The data obtained indicate unambigously that s4U residue contacts with tRNA (adenine-1-)-methyltransferase within the corresponding specific complex.

Biochimie, 1987 Oct, 69(10), 1097 - 104
An unusual rRNA operon constellation: in Thermus thermophilus HB8 the 23S/5S rRNA operon is a separate entity from the 16S rRNA operon; Hartmann RK et al.; We succeeded in identifying a promoter element within 200 base pairs upstream a transcriptional unit comprising only a 23S rRNA, 5S rRNA and a tRNA(gly) gene in Thermus thermophilus HB8 {1, 2} . This element shows a high degree of homology to the -35 and -10 consensus sequences for promoters described for Escherichia coli {3, 4} . The promoter activity was measured by the induction of the synthesis of functional chloramphenicol acetyltransferase in Escherichia coli . A region located at the transcriptional start, rich in guanosines and cytidines, is very similar in sequence to the one believed to be under stringent control in stable RNA and ribosomal protein genes of Escherichia coli {5} . Employing nuclease S1 protection we were able to determine the in vivo start of transcription, which was identical with the in vitro start using Escherichia coli RNA-polymerase . Furthermore we identified sequences in the region following the origin of transcription, which are homologous to sections in Escherichia coli rrn promoter-leader regions responsible for antitermination . Our finding of a promoter immediately preceding a 23S/5S rRNA operon proves a transcriptional decoupling of the 16S rRNA genes, a situation so far unprecedented among prokaryotes.

Biol Chem Hoppe Seyler, 1987 Oct, 368(10), 1391 - 9
Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria . VII . Nucleotide sequence of the lactate dehydrogenase gene from the mesophilic bacterium Bacillus megaterium . Preparation and properties of a hybrid lactate dehydrogenase comprising moieties of the B . megaterium and B . stearothermophilus enzymes; Waldvogel S et al.; The lactate dehydrogenase (LDH) gene of a mesophilic bacterium, Bacillus megaterium (DSM 090), was cloned in E . coli HB 101 using a pEMBL vector and synthetic oligonucleotide probes . The gene was strongly expressed in the vector used if the orientation of the insert allowed the LDH promoter and the vector's lac promoter to direct transcription in the same direction . The gene and its 5' and 3' flanking regions have been sequenced . Codon usage patterns of LDH genes from mesophilic and thermophilic bacilli were compared and found to be characteristically different . A hybrid gene was constructed from fragments of the LDH genes from B . stearothermophilus (coding for aa 15-100) and B . megaterium (coding for aa 101-331) . The hybrid LDH, named S100M, was more thermostable than B . megaterium LDH, less thermostabile than B . stearothermophilus LDH and unlike the two wildtype enzymes, it could not be activated by Fru-P2.

J Bacteriol, 1987 Oct, 169(10), 4857 - 60
Structure and organization of the hisA gene of the thermophilic archaebacterium Methanococcus thermolithotrophicus; Weil CF et al.; A restriction fragment of Methanococcus thermolithotrophicus genomic DNA was cloned into pUC8 to produce plasmid pET9301, which complements mutations in the hisA gene of Escherichia coli . Sequencing the DNA (2,155 base pairs) cloned from this thermophilic methanogen demonstrated that the M . thermolithotrophicus hisA gene is located within a cluster of open reading frames (ORFs) and is 68 and 69% homologous at the nucleotide level to the hisA genes of the mesophilic methanococci M . voltae and M . vannielii, respectively . The ORF (ORF 206) immediately 5' to the hisA gene of M . thermolithotrophicus is partially deleted in the genomes of the two mesophilic species, whereas ORF 114, which is 5' to ORF 206, is conserved in all three species.

J Biochem (Tokyo), 1987 Oct, 102(4), 875 - 83
Single-site catalysis of F1-ATPase from thermophilic bacterium PS3 and its dominance in steady-state catalysis at low ATP concentration; Yohda M et al.; Single-site catalysis by F1-ATPase from a thermophilic bacterium PS3 (TF1) was examined by incubating the enzyme with a submolar amount of radioactive ATP . The profile of single-site catalysis by TF1 at 23 degrees C was different from that of beef heart mitochondrial F1-ATPase (MF1) . ATP hydrolysis on the enzyme and release of the products was rapid, and subsequent addition of non-radioactive ATP (cold chase) did not promote the hydrolysis of radioactive ATP, indicating that the rate-limiting step was not the step of product release but the step of ATP binding to the enzyme . Thus, the characteristic features of so-called uni-site catalysis were not observed . At 60 degrees C, whether in the presence or absence of phosphate ion, a small amount of bound {alpha, gamma-32P}ATP and cold chase promotion were observed . However, since bound 32P1 was not detected by centrifugal gel filtration, it is not yet certain whether TF1 has typical uni-site characteristics . Based on the hydrolytic turnover rate for single-site catalysis and analysis of the kinetics of steady-state catalysis, it is proposed that single-site catalysis is dominant even in steady-state catalysis at ATP concentrations of less than about 20 microM.

Biochimie, 1987 Oct, 69(10), 1105 - 12
Covalent cross-linking of AcVal-tRNA to Tetrahymena thermophila cytoplasmic ribosomes and two of its 17S rRNA mutants; Nurse K et al.; Tetrahymena thermophila 80S ribosomes have been cross-linked to non-enzymatically bound AcVal-tRNA, presumably at the ribosomal P-site . Like the ribosomes from Escherichia coli, yeast, and Artemia salina, cross-linking is exclusively to C-1609, the equivalent of the E . coli C-1400 residue . Mutation of the RNA from G-1707 to A or from U-1711 to C which results in resistance to paromomycin or hygromycin, respectively, failed to affect the rate, yield, or site of cross-linking . The presence of the antibiotics during cross-linking also was without effect . It is concluded that at these two positions the base changes made do not interfere with the tertiary structure of the decoding site.

Biochim Biophys Acta, 1987 Sep 24, 915(2), 225 - 37
Structural and spectroscopic comparison of manganese-containing superoxide dismutases; Bjerrum MJ; Predicted secondary structures and optical properties of four manganese-containing superoxide dismutases isolated from Saccharomyces cerevisiae, Bacillus stearothermophilus, Escherichia coli and human liver are compared . The structural predictions are further compared with the known crystal structure of the manganese-containing superoxide dismutase from Thermus thermophilus HB8 . The secondary structures of the four dismutases are predicted by the methods of Chou and Fasman (Adv . Enzymol . 47 (1978) 45-148), Garnier et al . (J . Mol . Biol . 120 (1978) 97-120) and Lim (J . Mol . Biol . 88 (1974) 873-894) . The three models show satisfactory agreement and predict that the enzymes have a mixed alpha-helix and beta-sheet structure, and that they have homologous structures . The former conclusion is also reached from an analysis of the hydrophobic character of the amino-acid sequences of the four proteins according to Kyte and Doolittle (J . Mol . Biol . 157 (1982) 105-132) . The calculation of the secondary structure based on the 185-260 nm circular dichroism spectrum of manganese-containing superoxide dismutase from S . cerevisiae reveals that the enzyme consists of 61% alpha-helix, 13% beta-sheet, 11% turn and 8% random coil conformations, which is in good accordance with the prediction based on the amino-acid sequences . Comparison of the 400-700 nm circular dichroism spectra of manganese-containing superoxide dismutase from S . cerevisiae, E . coli and T . thermophilus demonstrates that manganese atoms have homologous coordination in the three enzymes . This investigation based on primary structures and spectral properties indicates that the four dismutases have the same overall structure . Since the structural predictions are in good agreement with the structure found for the manganese-containing superoxide dismutase from T . thermophilus HB8, it can be concluded that this structure is representative for the four enzymes and probably for manganese-containing superoxide dismutases in general.

Eur J Biochem, 1987 Sep 15, 167(3), 595 - 600
The photochemical reaction center of Chloroflexus aurantiacus is composed of two structurally similar polypeptides; Shiozawa JA et al.; A method has been devised which allowed the isolation of highly purified reaction center from the thermophilic green bacterium, Chloroflexus aurantiacus . The procedure consisted of three chromatography steps . The final step was fast protein liquid chromatography on Mono Q in the presence of nonanoyl-N-methylglucamide (Mega-9) . The purified reaction center complex was photochemically active and had an A280/A813 of 1.4 or less . Under non-denaturing conditions, a pigmented protein band having a Mr of 52,000-55,000 was observed in sodium dodecyl sulfate gels . When the isolated complex was heat-dissociated in the presence of sodium dodecyl sulfate, just two polypeptides having very similar Mr (24,000 and 24,500) were observed . Two protein bands were also observed in two-dimensional isoelectric focusing/sodium-dodecyl-sulfate polyacrylamide gel electrophoresis; the PI values of the two polypeptides were 6.5 and 6.7 . Partial peptide mapping of the two isolated subunits, using both enzymatic and chemical cleavage techniques, yielded almost identical patterns which indicated a high degree of sequence homology between the two polypeptides . The N-terminal amino acid sequences of the two polypeptides were identical and did not exhibit any homology to reaction center subunits of purple sulfur bacteria . The Chloroflexus reaction center is believed to be composed of one molecule of each polypeptide, the photoactive bacteriochlorophyll a dimer and, as accessory pigments, an additional bacteriochlorophyll a and three bacteriopheophytins . Hence, it appears to be the smallest photochemically active reaction center isolated to date.

Eur J Biochem, 1987 Sep 15, 167(3), 475 - 9
A novel archaebacterial NAD+-dependent alcohol dehydrogenase . Purification and properties; Rella R et al.; An NAD+-dependent alcohol dehydrogenase (alcohol: NAD+ oxidoreductase, EC 1.1.1.1) was detected in cellular extracts of the extreme thermophilic archaebacterium Sulfolobus solfataricus . The enzyme was purified to homogeneity and shown to be a dimer with a native molecular mass of 71 kDa by sucrose gradient centrifugation and SDS electrophoresis . The enzyme has a broad substrate specificity that includes linear and branched primary alcohols, linear and cyclic secondary alcohols, linear and cyclic ketones and anisaldehyde . The enzyme has an extraordinary thermophilicity and a remarkable thermostability, and appears to have some properties and a structure different from those previously described for thermophilic alcohol dehydrogenases.

Cell, 1987 Sep 11, 50(6), 951 - 61
Selection of circularization sites in a group I IVS RNA requires multiple alignments of an internal template-like sequence; Been MD et al.; Circularization and reverse circularization of the Tetrahymena thermophila rRNA intervening sequence resemble the first and second steps in splicing, respectively . However, site-specific base substitutions show that different nucleotides are involved in selection of the 5' splice site and the circularization sites . Furthermore, a substitution at the major circularization site that prevents circularization can be suppressed by second substitutions at two different nucleotide positions . A model is proposed in which adjacent and overlapping sequences can function as a binding site, forming a short duplex with the sequence at the circularization site and thus directing circularization and reverse circularization . Because the 5' exon-binding site and three potential circularization binding sites fall within a contiguous eight nucleotide region, this sequence may translocate relative to the catalytic core of the ribozyme in a template-like manner.

J Biol Chem, 1987 Sep 5, 262(25), 11979 - 81
A new naturally occurring polyamine containing a quaternary ammonium nitrogen; Oshima T et al.; A new polyamine, tetrakis(3-aminopropyl)ammonium, N+ (CH2CH2CH2NH2)4, was identified in cells of an extreme thermophile, Thermus thermophilus . This compound was chemically synthesized and its chemical properties were coincident with those of the amine isolated from the thermophile.

Biochim Biophys Acta, 1987 Sep 4, 921(1), 7 - 12
Pathway of taurolipid B formation from exogenous taurolipid A by Tetrahymena thermophila; Kaya K et al.; When Tetrahymena thermophila was incubated with taurolipid A isolated from T . pyriformis NT-1, the exogenously added taurolipid A was deacylated rapidly, and taurolipid B content in the cells was increased . The deacylated taurolipid A (lysotaurolipid A) content reached a maximum early on during incubation, and then declined . Taurolipid B and lysotaurolipid B contents in the cells were increased continuously during the incubation . These observations suggest that lysotaurolipid A was an intermediate for taurolipid B formation . When cells were incubated with lysotaurolipid A, newly formed lysotaurolipid B and taurolipid B were observed . Furthermore, when cells were incubated with lysotaurolipid B, only taurolipid B was newly formed . In contrast, newly formed lysotaurolipid B was observed when cells were incubated with exogenous taurolipid B . From the results, we have postulated the biosynthetic pathway of taurolipid B from exogenous taurolipid A in cells of Thermophila.

Equine Vet J, 1987 Sep, 19(5), 442 - 7
Technique for assessing respiratory health hazards from hay and other source materials; Clarke AF et al.; This paper describes and compares three techniques of categorisation of hay, straw and other feeds and beddings collected from stables . A hand-held sampler was used to categorise samples according to the presence of plant material, fungal spores and dust mites . An Andersen sampler was used to categorise samples according to the thermotolerances of fungi and actinomycetes . An aerodynamic particle sizer was used to categorise samples according to respirable particle release rates . The highest burden of respirable particles was associated with the presence of thermophilic and thermotolerant actinomycetes and fungi . The portable slit sampler proved to be an accurate, quick and simple semiquantitative method of assessing the mould contamination of source materials . This latter technique requires only a microscope and the sampler, and is thus ideal for veterinary practices and small diagnostic laboratories.

Biol Chem Hoppe Seyler, 1987 Sep, 368(9), 1167 - 77
Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria, VI . Nucleotide sequences of lactate dehydrogenase genes from the thermophilic bacteria Bacillus stearothermophilus, B . caldolyticus and B . caldotenax; Zulli F et al.; Based on the previously determined amino-acid sequence of lactate dehydrogenase from B . stearothermophilus, an oligonucleotide probe was synthesized and used to clone the structural genes for lactate dehydrogenase from B . stearothermophilus, B . caldolyticus and B . caldotenax . The nucleotide sequences of the entire LDH genes from these three thermophilic bacilli were determined by the method of Maxam and Gilbert . The nucleotide sequence of the LDH gene from B . stearothermophilus is exactly identical to the one published recently; it agrees with the experimentally determined amino-acid sequence except at three positions . The amino-acid homologies among these thermophilic enzymes are 90% or more . The LDH genes are efficiently expressed in E . coli.

Appl Environ Microbiol, 1987 Sep, 53(9), 2111 - 8
Enzymatic methylation of sulfide, selenide, and organic thiols by Tetrahymena thermophila; Drotar A et al.; Cell extracts from the ciliate Tetrahymena thermophila catalyzed the S-adenosylmethionine-dependent methylation of sulfide . The product of the reaction, methanethiol, was detected by a radiometric assay and by a gas-chromatographic assay coupled to a sulfur-selective chemiluminescence detector . Extracts also catalyzed the methylation of selenide, and the product was shown by gas chromatography-mass spectrometry to be methaneselenol . The sulfide and selenide methyltransferase activities copurified with the aromatic thiol methyltransferase previously characterized from this organism (A.-M . Drotar and R . Fall, Pestic . Biochem . Physiol . 25:396-406, 1986), but heat inactivation experiments suggested the involvement of distinct sulfide and selenide methyltransferases . Short-term toxicity tests were carried out for sulfide, selenide, and their methylated derivatives; the monomethylated forms were somewhat more toxic than the nonmethylated or dimethylated compounds . Cell suspensions of T . thermophila exposed to sulfide, methanethiol, or their selenium analogs emitted methylated derivatives into the headspace . These results suggest that this freshwater protozoan is capable of the stepwise methylation of sulfide and selenide, leading to the release of volatile methylated sulfur or selenium gases.

Genetics, 1987 Sep, 117(1), 13 - 23
Nucleo-cytoplasmic interaction during macronuclear differentiation in ciliate protists: genetic basis for cytoplasmic control of SerH expression during macronuclear development in Tetrahymena thermophila; Doerder FP et al.; A novel class of mutations affecting the developmental expression of SerH cell surface antigen genes of Tetrahymena thermophila is described . Unlike previous categories of mutation, the four independently isolated mutations of this class act through the cytoplasm to affect SerH genes during macronuclear development . That is, macronuclei which develop under the influence of mutant cytoplasm do not subsequently express H, most likely because the developmental processing of SerH genes is affected . The cytoplasmic effect is specific for the SerH locus and is independent of which SerH allele is present . In place of H, hitherto unknown antigens are expressed . Expression of SerH can be rescued during development either by wild-type cytoplasm exchanged between conjugants or by the homozygous wild-type genotype . The mutations segregate independently of the SerH genes and identify one, possibly two, bistable genes . Possible models to explain these results are discussed.

Acta Paediatr Scand, 1987 Sep, 76(5), 754 - 62
Acute gastroenteritis in children attending day-care centres with special reference to rotavirus infections . I . Aetiology and epidemiologic aspects; Hjelt K et al.; Acute gastroenteritis (GE) among 214 children (aged 6 months-7 years) attending day-care centres (DDCs) in the Copenhagen County was studied during a 12-month period . A total of 197 cases of GE was observed in 109 children (i.e . 51% of the participants) . The aetiology was as follows: rotavirus (n = 48) (24%), pathogenic bacteria (n = 11) (6%), Giardia lamblia (n = 3) (2%), while the aetiology of 68% remains unknown . The pathogenic bacteria included Yersinia enterocolitica, thermophilic Campylobacter, Clostridium difficile (+/- toxin) and enteropathogenic E . coli . In 4% of the GE the infections were multiple and Cryptosporidium was seen in one of these cases . The rate of GE declined with age from 1.35 GE per child per year (age group 1.0- less than 2.0 years) to 0.36 (6.0- less than 8.0 years) . Serum sampled at the start of the study period showed that the frequency of detectable rotavirus IgG increased with age from 48% in the 6 months- less than 1.0 year group to 96% in the 4.0- less than 7.0 year group . The highest rates of rotavirus GE occurred from January to April (i.e . the rotavirus season) . Moreover, rotavirus GE was almost absent after the age of 4 . Hence, the rates of rotavirus GE per rotavirus season per child were 0.80 (age group 6 months-less than 1.0 year), 0.32 (1.0-less than 2.0), 0.14 (2.0-less than 3.0), 0.16 (3.0-less than 4.0), 0.06 (4.0-less than 5.0) and 0.04 (5.0-less than 6.0) . Only 2 out of the 48 rotavirus GE were reinfections.(ABSTRACT TRUNCATED AT 250 WORDS)

J Clin Microbiol, 1987 Sep, 25(9), 1747 - 52
Prevalence and characterization of hippurate-negative Campylobacter jejuni in King County, Washington; Totten PA et al.; A total of 593 strains of thermophilic Campylobacter species were isolated either from humans with diarrhea or from poultry in King County, Washington . Of these strains, 98 (52 hippurate-positive strains and all 46 of the hippurate-negative strains) were selected for further phenotypic characterization and genetic classification . Hippurate hydrolysis, the test typically used to differentiate Campylobacter jejuni and C . coli, did not always correlate with the genetic classification . All hippurate-positive strains were classified as C . jejuni . Of the hippurate-negative strains, 20% were C . jejuni, 78% were C . coli, and 2% were C . laridis . Assuming that the remaining hippurate-positive strains were all C . jejuni, then hippurate-negative C . jejuni represented a small percentage (9 of 556 or 1.6%) of C . jejuni strains but a significant percentage (9 of 46 or 20%) of hippurate-negative strains . This finding suggests that hippurate hydrolysis should not be used as the sole criterion for differentiating thermophilic Campylobacter species, particularly when describing the disease states associated with these organisms.

Biochimie, 1987 Sep, 69(9), 975 - 82
5S RNA-protein complexes released by EDTA treatment of 60S ribosomal subunits of Tetrahymena thermophila; Hayes F et al.; Treatment of large (60S) subunit of the cytoplasmic ribosome of the protozoa Tetrahymena thermophila with EDTA causes quantitative release of 5S rRNA associated with variable non quantitative amounts of one or more of 60S proteins L4, L15, L24, L31 and L41 . The composition of the group of proteins released with 5S rRNA depends on both the molar ratio of EDTA and 60S subunits and the concentration of 60S subunits, in treatment mixtures.

Biol Chem Hoppe Seyler, 1987 Sep, 368(9), 1157 - 66
Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria, V . The complete amino-acid sequence of the mesophilic L-lactate dehydrogenase from Bacillus megaterium; Stangl D et al.; The complete amino-acid sequence of L-lactate-dehydrogenase from the mesophilic Bacillus megaterium was determined . 92% of the 318 amino acids were established by sequence analysis of the N-terminus, of four CNBr fragments and of one fragment obtained by cleavage with BNPS-skatole . The primary structure was completed by sequencing overlapping fragments obtained by further cleavage of suitable CNBr fragments and BNPS fragments with either trypsin, endoproteinase Lys-C, o-iodosobenzoic acid or hydroxylamine . The C-terminal amino acids were determined by degradation with carboxypeptidase A . The sequence homology between lactate dehydrogenases from B . megaterium and those from other Bacilli is 59-61% and 35-37% to those from higher organisms . The high sequence homology among lactate dehydrogenases from Bacilli, adapted to different temperatures, allows comparative studies of the structural basis of protein thermostability.

Appl Environ Microbiol, 1987 Sep, 53(9), 2165 - 70
Antimicrobial activity of lysozyme against bacteria involved in food spoilage and food-borne disease; Hughey VL et al.; Egg white lysozyme was demonstrated to have antibacterial activity against organisms of concern in food safety, including Listeria monocytogenes and certain strains of Clostridium botulinum . We also found that the food spoilage thermophile Clostridium thermosaccharolyticum was highly susceptible to lysozyme and confirmed that the spoilage organisms Bacillus stearothermophilus and Clostridium tyrobutyricum were also extremely sensitive . Several gram-positive and gram-negative pathogens isolated from food poisoning outbreaks, including Bacillus cereus, Clostridium perfringens, Staphylococcus aureus, Campylobacter jejuni, Escherichia coli O157:H7, Salmonella typhimurium, and Yersinia enterocolitica, were all resistant . The results of this study suggest that lysozyme may have selected applications in food preservation, especially when thermophilic sporeformers are problems, and as a safeguard against food poisoning caused by C . botulinum and L . monocytogenes.

J Bacteriol, 1987 Sep, 169(9), 4302 - 7
Cloning of the debranching-enzyme gene from Thermoanaerobium brockii into Escherichia coli and Bacillus subtilis; Coleman RD et al.; The gene for an enzyme with single or dual specificity on complex carbohydrates has been transferred from its native host (Thermoanaerobium brockii), a thermophilic anaerobe, into Escherichia coli and Bacillus subtilis . Most of the gene coding region is in a 2.2-kilobase PstI fragment that is common to the E . coli and B . subtilis chimeric vectors pCPC902 and pCPC903, respectively . Although the T . brockii debranching enzyme secreted from B . subtilis was unglycosylated and had less thermostability, more enzyme was secreted from B . subtilis (0.80 to 1.0 U/ml) than from T . brockii (0.23 U/ml) . E . coli did not export any measurable enzyme . From the fermentation broth of B . subtilis containing pCPC903, three active species of the debranching enzyme were separated; two species are possibly protease digestion products of the larger protein (105,000 molecular weight) . Whereas the enzyme can cleave all of the alpha-1----6 glucosidic linkages (and none of the alpha-1----4 bonds) in pullulan, it hydrolyzed mostly alpha-1----4 and very few of the alpha-1----6 linkages in starch . Upon hydrolysis of pullulan by the enzyme, only maltotriose was produced, while starch was digested to various-sized oligomers.

Appl Environ Microbiol, 1987 Sep, 53(9), 2077 - 81
Inactivation of animal viruses during sewage sludge treatment; Spillmann SK et al.; Using a previously developed filter adsorption technique, the inactivation of a human rotavirus, a coxsackievirus B5, and a bovine parvovirus was monitored during sludge treatment processes . During conventional anaerobic mesophilic digestion at 35 to 36 degrees C, only minor inactivation of all three viruses occurred . The k' values measured were 0.314 log10 unit/day for rotavirus, 0.475 log10 unit/day for coxsackievirus B5, and 0.944 log10 unit/day for parvovirus . However, anaerobic thermophilic digestion at 54 to 56 degrees C led to rapid inactivation of rotavirus (k' greater than 8.5 log10 units/h) and of coxsackievirus B5 (k' greater than 0.93 log10 unit/min) . Similarly, aerobic thermophilic fermentation at 60 to 61 degrees C rapidly inactivated rotavirus (k' = 0.75 log10 unit/min) and coxsackievirus B5 (k' greater than 1.67 log10 units/min) . Infectivity of parvovirus, however, was only reduced by 0.213 log10 unit/h during anaerobic thermophilic digestion and by 0.353 log10 unit/h during aerobic thermophilic fermentation . Furthermore, pasteurization at 70 degrees C for 30 min inactivated the parvovirus by 0.72 log10 unit/30 min . In all experiments the contribution of temperature to the total inactivation was determined separately and was found to be predominant at process temperatures above 54 degrees C . In conclusion, the most favorable treatment to render sludge hygienically safe from the virological point of view would be a thermal treatment (60 degrees C) to inactivate thermolabile viruses, followed by an anaerobic mesophilic digestion to eliminate thermostable viruses that are more sensitive to chemical and microbial inactivations.

J Biol Chem, 1987 Aug 15, 262(23), 11088 - 96
31P and 13C NMR analyses of the energy metabolism of the thermophilic anaerobe Clostridium thermocellum; Tolman CJ et al.; The energy metabolism of an anaerobic obligate thermophile, Clostridium thermocellum, has been examined as a function of incubation temperature using 31P NMR spectroscopy . Specifically investigated were the generation and availability of ATP as a function of temperature, activation energies for key processes in energy metabolism including formation of a pH gradient across the cell membrane, transport of key nutrients, and initial steps in glycolysis, and the existence of a membrane phase transition in the intact organism . Cells generate ATP via glycolysis at all temperatures examined; hence, limitation of the energy supply is not directly responsible for the lack of growth of this organism at low temperatures . Estimations of activation energies show a distinct hierarchy in the ATP-utilizing reactions examined . Conservation of ATP hydrolysis energy as delta pH has the lowest activation energy (less than or equal to 4 kcal/mol), two transport processes exhibit 10 kcal/mol activation energies, and early phosphorylation steps in glycolysis have significantly higher activation energies (approximately 25 kcal/mol) . Neither the membrane-bound ATPase responsible for formation of the pH gradient nor the permease involved in phosphate transport shows evidence of a change in behavior around the phase transition temperature determined for extracted lipids of C . thermocellum . Line widths of inorganic phosphate do show a break in behavior around 35-40 degrees C . Possible explanations for this behavior are discussed.

J Mol Biol, 1987 Aug 5, 196(3), 517 - 24
Identification of a vegetative promoter in Myxococcus xanthus . A protein that has homology to histones; Komano T et al.; A physical map of 330 x 10(3) base-pairs near the replication origin of Myxococcus xanthus chromosome has been established already . Using DNA fragments from this region, Northern blot hybridization analysis was carried out in order to identify the genes expressed during vegetative growth . One of the genes, tentatively designated as vegA, was cloned and its entire DNA sequence was determined . The amino acid sequence of the gene product deduced from the DNA sequence reveals that the VegA protein is a very basic protein with a molecular weight of 18,700 . The gene was expressed in Escherichia coli using an expression vector, and its gene product was identified using SDS/polyacrylamide gel electrophoresis . From the results of S1 nuclease mapping, the vegA promoter was found to contain the sequence TAGACA at the -35 region and the sequence AAGGGT at the -10 region . These two regions are separated by 18 nucleotides . Genetic analysis suggests that the vegA gene may be essential for the growth of M . xanthus . From a computer-aided search for homologies to know protein structures, it was found that the VegA protein has homologies to histone H4 of Tetrahymena thermophila and histone H2B of sea urchin.

J Cell Sci, 1987 Aug, 88 ( Pt 1), 47 - 55
Isolation and characterization of a mutant of Tetrahymena thermophila blocked in secretion of lysosomal enzymes; Hunseler P et al.; The development of a sensitive screening procedure for mutants of Tetrahymena thermophila blocked in secretion of lysosomal enzymes is described . By means of this procedure a mutant blocked in secretion of lysosomal enzymes has been isolated . This sec- mutant, MS-1, is constitutively blocked in release of at least six lysosomal enzymes, under both nutrient and non-nutrient conditions . MS-1 possesses, bound within the cell, the same amount of active lysosomal enzymes as the wild type . During starvation in media of low ionic strength MS-1 develops a highly vacuolated phenotype . This phenotype is caused by the sec- allele . It is reversed to a normal cell shape when the mutant is transferred to isotonic medium . The sec- mutant MS-1 contains mucocysts and is capable of inducing exocytosis of these secretory organelles, suggesting that Tetrahymena possesses at least two independent protein-secreting organelles.

Zentralbl Bakteriol Mikrobiol Hyg {A}, 1987 Aug, 266(1-2), 94 - 103
Occurrence, serotypes and biotypes of thermophilic Campylobacters isolated in Vienna; Hirschl AM et al.; During the 1982-1986 period of all bacterial pathogens found to have caused diarrhoea, 35% belonged to the genus Campylobacter (C) . Approximately 70% of the strains were isolated from persons under the age of 30 years, with a distinct peak of occurrence in the autumn . Biotyping and serotyping according to Lior yielded the following results: C . jejuni biotype I: 32.9%, C . jejuni biotype II: 48.6%, C . coli biotype I: 10.3%, C . coli biotype II: 8.2% . From the 121 strains serotyped, 118 (97.5%) were typable . The serotypes most frequently encountered were type 1 (15.7%), 4 (9.9%), 2 and 11 (7.4% each) . There were 2 familial outbreaks of Campylobacter enteritis which could be completely elucidated by biotyping and serotyping . One outbreak was caused by C . jejuni biotype I serotype 11, the other by C . jejuni biotype II serotype 6 . Considering the frequent occurrence of Campylobacter infections, isolates should be routinely typed . The existing typing methods and schemes are highly developed.

J Bacteriol, 1987 Aug, 169(8), 3792 - 800
Specialized cell surface structures in cellulolytic bacteria; Lamed R et al.; The cell surface topology of various gram-negative and -positive, anaerobic and aerobic, mesophilic and thermophilic, cellulolytic and noncellulolytic bacteria was investigated by scanning electron microscopic visualization using cationized ferritin . Characteristic protuberant structures were observed on cells of all cellulolytic strains . These structures appeared to be directly related to the previously described exocellular cellulase-containing polycellulosomes of Clostridium thermocellum YS (E . A . Bayer and R . Lamed, J . Bacteriol . 167:828-836, 1986) . Immunochemical evidence and lectin-binding studies suggested a further correlation on the molecular level among cellulolytic bacteria . The results indicate that such cell surface cellulase-containing structures may be of general consequence to the bacterial interaction with and degradation of cellulose.

Cell, 1987 Jul 31, 50(3), 477 - 83
Dynamics of telomere length variation in Tetrahymena thermophila; Larson DD et al.; We have analyzed the mechanism and dynamics of telomere length variation in the macronucleus of Tetrahymena thermophila . In a newly differentiated macronucleus, the average length of the telomeric repeated sequence, (C4A2 X T2G4)n, is closely regulated . In contrast, in vegetatively dividing cells in log phase, all macronuclear telomeric sequences lengthen coordinately by 3-10 bp per generation until up to 1000 bp are added . In both elongated and short telomeres, characteristic single-stranded breaks on both strands are distally located . Reduction of elongated telomeres to their original length involves either the appearance of a novel type of variant cell, incapable of net telomere elongation, or, under stationary phase conditions, a reversible removal of telomeric sequences . The demonstration that telomeres are dynamic structures provides evidence for a model of telomere length regulation by activities that add and remove telomeric repeats.

Biochem Biophys Res Commun, 1987 Jul 31, 146(2), 705 - 10
In vitro mutated beta subunits from the F1-ATPase of the thermophilic bacterium, PS3, containing glutamine in place of glutamic acid in positions 190 or 201 assembles with the alpha and gamma subunits to produce inactive complexes; Ohtsubo M et al.; Using site-directed mutagenesis, Glu-190 or Glu-201 of the beta subunit of the F1-ATPase from the thermophilic bacterium PS3 were replaced with glutamine . It was possible to reconstitute complexes of the mutated beta subunits with alpha and gamma subunits, but the complexes did not have ATPase activity . It is concluded that carboxylic acid side chains of Glu-190 and Glu-201 of the beta subunit are essential for catalytic activity of F1-ATPase.

Nucleic Acids Res, 1987 Jul 24, 15(14), 5681 - 97
Characterization of two types of histone H2B genes from macronuclei of Tetrahymena thermophila; Nomoto M et al.; Two histone H2B gene clones were isolated from macronuclei of Tetrahymena thermophila . Nucleotide sequences of the two clones were highly homologous within the coding region but not in the noncoding region . Comparison of the deduced amino acid sequences between the two clones showed three differences in a total of 121 amino acids . Each of the two clones contained a TAA triplet within the coding region, which appeared to code for a glutamine residue . To demonstrate the existence of histone mRNA containing UAA triplet, nuclease P1 protection mapping using total cellular RNA and nucleotide sequencing of primer extension products were carried out . The results clearly indicated that two cloned histone H2B genes were transcribed, giving rise to the major histone H2B mRNAs with a UAA triplet sequence in frame . The tentative 5'- and 3'-ends of histone H2B mRNAs were determined.

J Mol Biol, 1987 Jul 20, 196(2), 441 - 2
Preliminary X-ray data for a D-amino acid amino-transferase from a novel thermophilic Bacillus; Stoddard B et al.; Crystals of the D-amino acid aminotransferase (D-ATA) from a novel thermophilic Bacillus species (Escherichia coli pICT113 cloned gene product) have been examined by X-ray analysis . The crystals grow as hexagonal prisms, with the symmetry of space group P61 or P65 (indistinguishable crystallographically) . The cell dimensions are a = b = 135 A, c = 53 A, alpha = beta = 90 degrees, and gamma = 120 degrees . The unit cell has a volume of 850,000 A3 with six asymmetric units per unit cell . There is one dimer of molecular weight 62,000 per asymmetric unit, and the crystals diffract to 2.7 A.

J Mol Biol, 1987 Jul 5, 196(1), 217 - 21
Origin of the phosphate at the ligation junction produced by self-splicing of Tetrahymena thermophila pre-ribosomal RNA; Price JV; The exons of the self-splicing pre-ribosomal RNA of Tetrahymena thermophila are joined accurately in vitro, even when only 33 nucleotides of the natural 5' exon and 38 nucleotides of the natural 3' exon remain . RNA fingerprint analysis was used to identify the unique ribonuclease T1 oligonucleotide generated by exon ligation . Secondary digests of the ligation junction oligonucleotide with ribonuclease A confirmed the identity of the fragment and demonstrated that the phosphate group that forms the phosphodiester bond at the ligation junction is derived from the 5' position of a uridine nucleotide in the RNA . This observation supports the prediction that the splice junction phosphate is derived from the 3' splice site . These results emphasize the mechanistic similarities of RNA splicing reactions of the group I introns, group II introns and nuclear pre-mRNA introns.

J Mol Biol, 1987 Jul 5, 196(1), 49 - 60
5' exon requirement for self-splicing of the Tetrahymena thermophila pre-ribosomal RNA and identification of a cryptic 5' splice site in the 3' exon; Price JV et al.; The intervening sequence (IVS) of the Tetrahymena thermophila ribosomal RNA precursor undergoes accurate self-splicing in vitro . The work presented here examines the requirement for Tetrahymena rRNA sequences in the 5' exon for the accuracy and efficiency of splicing . Three plasmids were constructed with nine, four and two nucleotides of the natural 5' exon sequence, followed by the IVS and 26 nucleotides of the Tetrahymena 3' exon . RNA was transcribed from these plasmids in vitro and tested for self-splicing activity . The efficiency of splicing, as measured by the production of ligated exons, is reduced as the natural 5' exon sequence is replaced with plasmid sequences . Accurate splicing persists even when only four nucleotides of the natural 5' exon sequence remain . When only two nucleotides of the natural exon remain, no ligated exons are observed . As the efficiency of the normal reaction diminishes, novel RNA species are produced in increasing amounts . The novel RNA species were examined and found to be products of aberrant reactions of the precursor RNA . Two of these aberrant reactions involve auto-addition of GTP to sites six nucleotides and 52 nucleotides downstream from the 3' splice site . The former site occurs just after the sequence GGU, and may indicate the existence of a GGU-binding site within the IVS RNA . The latter site follows the sequence CUCU, which is identical with the four nucleotides preceding the 5' splice site . This observation led to a model where where the CUCU sequence in the 3' exon acts as a cryptic 5' splice site . The model predicted the existence of a circular RNA containing the first 52 nucleotides of the 3' exon . A small circular RNA was isolated and partially sequenced and found to support the model . So, a cryptic 5' splice site can function even if it is located downstream from the 3' splice site . Precursor RNA labeled at its 5' end, presumably by a GTP exchange reaction mediated by the IVS, is also described.

Mol Biol Evol, 1987 Jul, 4(4), 381 - 94
The evolution of prokaryotic ferredoxins--with a general method correcting for unobserved substitutions in less branched lineages; Fitch WM et al.; Thirty-one bacterial type ferredoxins were examined by means of the parsimony method for their phylogenetic implications . The results show reasonable relationships in that photosynthetic, thermophilic, and desulfovibrio groups are identifiable; but a number of interesting anomalies occur . These include a methanogen sequence that clusters among the desulfovibrios . There are several differences from the phylogeny of Woese . At least two duplications producing paralogous genes are demonstrated, plus the probable existence of two more . The partial internal gene duplication that doubled the length of ferredoxin is confirmed by showing that the probability of the two ancestrally reconstructed halves possessing that much similarity by chance is 10(-7) . Howard and co-workers proposed that the two halves of the Azotobacter vinelandii are reversed relative to most other sequences . A phylogeny, drawn with the halves of the azotobacter sequence (and its relatives) reversed produced a tree that had only three less nucleotide substitutions than did the tree without their halves reversed . This plus other evidence suggests that the significantly greater similarity observed across rather than within the halves is more likely the result of convergence.

Mol Gen Genet, 1987 Jul, 208(3), 537 - 41
Synthesis and secretion of a heat-stable carboxymethylcellulose from Clostridium thermocellum in Bacillus subtilis and Bacillus stearothermophilus; Soutschek-Bauer E et al.; The cellulase gene celA of Clostridium thermocellum coding for the thermostable endoglucanase A was transferred from Escherichia coli to Bacillus subtilis 168 and B . stearothermophilus CU21 using plasmids derived from the Bacillus vector pUB110 . When the structural part of the gene was joined to a pUB110 promoter the recombinant plasmids (pSE102, pSE105) were stably maintained and expressed carboxymethylcellulose (CMCase) activity . In B . stearothermophilus CU21 (pSE105) the clostridial CMCase was produced over a wide temperature range up to the maximal growth temperature (68 degrees C) . In contrast to E . coli, all of the CMCase synthesized in bacilli was released into the culture medium . About 50% of the extracellular protein secreted by B . subtilis 168 (pSE102) carrying the celA gene consisted of endoglucanase A . These findings demonstrate the feasibility of producing cellulolytic enzymes from thermophilic anaerobes in bacilli.

Anal Biochem, 1987 Jul, 164(1), 72 - 7
Activity staining of cellulases in polyacrylamide gels containing mixed linkage beta-glucans; Schwarz WH et al.; Endoglucanase and cellobiohydrolase components of thermophilic cellulases can be detected in situ after gel electrophoresis in the presence of sodium dodecyl sulfate by incorporating a mixed linkage beta-glucan (barley beta-glucan, lichenan) in the separation gel . Zymograms are prepared after a renaturation treatment and incubation by staining the gel with Congo red . This method is suitable for the detection of beta-glucanases with different substrate specificities cleaving beta-1,4-, beta-1,4-1,3-, or beta-1,3-glucans . Cellobiohydrolase activities can be detected by adding 4-methylumbelliferyl-beta-D-cellobioside to the incubation buffer . The gels are subsequently stained with Coomassie blue to establish identical molecular weights of beta-glucanase and protein bands . Applications of this technique for the comparison of cellulases and for the identification of cellulase components expressed from recombinant clones are presented.

Nucleic Acids Res, 1987 Jun 25, 15(12), 4821 - 35
Gene organization, transcription signals and processing of the single ribosomal RNA operon of the archaebacterium Thermoproteus tenax; Kjems J et al.; The single ribosomal RNA (rRNA) operon from the extreme thermophile and archaebacterium Thermoproteus tenax was sequenced . Sites of transcriptional initiation and termination were established and the processing sites on the primary transcript were mapped with nuclease S1 . The operon contained genes coding for 16S and 23S RNAs but lacked those coding for tRNA and 5S RNA . Transcription initiates 175 bp upstream from the start of the 16S RNA gene (Wich et al., EMBO J . 6, 523-528, 1987) and terminates 49 bp downstream from the 23S RNA gene within a long pyrimidine sequence . An open reading frame downstream from the rRNA operon is transcribed . The sequences bordering both 16S and 23S RNA genes can form putative processing stems in the primary transcript that involve the whole of the 16S-23S RNA spacer . The stems contain irregular features that constitute processing signals and are conserved in other archaebacteria . The 16S RNA stem is cut prior to that of the 23S RNA and RNA maturation follows . An unusual 14 bp helix can form between the extremities of the transcript such that the whole transcript is highly structured and a fork-like structure is formed together with the processing stems . The 23S RNA sequence was aligned with other available 23S-like RNA sequences (Leffers et al., J . Mol . Biol . 195, in press): a putative secondary structure exhibiting archaebacterial-specific features was deduced using comparative sequence analyses . A rooted phylogenetic tree was also derived for the archaebacteria that confirms their division into three major subgroups.

Biochemistry, 1987 Jun 16, 26(12), 3330 - 40
Guanosine binding required for cyclization of the self-splicing intervening sequence ribonucleic acid from Tetrahymena thermophila; Tanner NK et al.; We have converted the intramolecular cyclization reaction of the self-splicing intervening sequence (IVS) ribonucleic acid (RNA) from Tetrahymena thermophila into an intermolecular guanosine addition reaction . This was accomplished by selectively removing the 3'-terminal nucleotide by oxidation and beta-elimination; the beta-eliminated IVS thereby is no longer capable of reacting with itself . However, under cyclization conditions, a free guanosine molecule can make a nucleophilic attack at the normal cyclization site . We have used this guanosine addition reaction as a model system for a Michaelis-Menten kinetic analysis of the guanosine binding site involved in cyclization . The results indicate that functional groups on the guanine that are used in a G-C Watson-Crick base pair are important for the cyclization reaction . This is the same result that was obtained for the guanosine binding site involved in splicing {Bass, B . L., & Cech, T . R . (1984) Nature (London) 308, 820-826} . Unlike splicing, however, certain additional nucleotides 5' to the guanosine moiety make significant binding contributions . We conclude that the guanosine binding site in cyclization is similar to, but not identical with, the guanosine binding site in splicing . The same binding interactions used in cyclization could help align the 3' splice site of the rRNA precursor for exon ligation . We also report that the phosphodiester bond at the cyclization site is susceptible to a pH-dependent hydrolysis reaction; the phosphodiester bond is somehow activated toward attack by the 3'hydroxyl of a guanosine molecule or by a hydroxyl ion.

Nucleic Acids Res, 1987 Jun 11, 15(11), 4583 - 91
The sequence of the 6S RNA gene of Pseudomonas aeruginosa; Vogel DW et al.; From the gram-negative eubacterium Pseudomonas aeruginosa we have isolated a stable 6S RNA, approximately 180 nucleotides in length . The RNA was partially sequenced and identified by comparison with the known Escherichia coli 6S RNA sequence . Southern hybridizations revealed a single copy gene coding for the 6S RNA . DNA from other prokaryotes, i.e . E . coli, Thermus thermophilus, Bacillus subtilis, Bacillus stearothermophilus and Halobacterium maris mortui, did not give detectable hybridization signals . The 6S RNA gene was cloned in E . coli and its complete primary structure was determined . Although the 6S RNA sequences from P . aeruginosa and E . coli share only a 60.4% homology, we are able to propose a common secondary structural model.

J Biol Chem, 1987 Jun 5, 262(16), 7523 - 7
Nucleotide sequence of the 18-25 S ribosomal RNA intergenic region from a thermophile, Thermomyces lanuginosus; Nazar RN et al.; To identify important structural features in the intergenic sequences of ribosomal DNAs, the nucleotide sequence of the 18-25 S rRNA intergene region was determined in a thermophilic fungus, Thermomyces lanuginosus, and compared to that of common yeasts . While a striking sequence homology was observed in the mature ribosomal sequences, the two internal transcribed spacer regions were found to be unusually G + C rich and significantly shorter than any other organism . An extensive secondary structure could be predicted but estimates of the secondary structure did not reveal a minimum structure which is common to other species; rather, in all rDNA transcripts the intergenic regions appear organized into a secondary structure in which all of the mature rRNA junctions are localized within one domain . This suggests that a role of the internal transcribed spacer in ribosome maturation may be that of a "biological spring" which maintains processed sites in close proximity.

Biochem J, 1987 Jun 1, 244(2), 331 - 5
Unusual ciliate-specific codons in Tetrahymena mRNAs are translated correctly in a rabbit reticulocyte lysate supplemented with a subcellular fraction from Tetrahymena; Andreasen PH et al.; The codon usage of Tetrahymena thermophila and other ciliates deviates from the 'universal genetic code' in that UAA and probably UAG are not translational termination signals but code for glutamine . Therefore, translation in vitro of mRNA from Tetrahymena in a reticulocyte lysate is prematurely terminated if a UAA or UAG triplet is present in the reading frame of the mRNA . We show that the addition of a subcellular fraction from Tetrahymena thermophila enables a rabbit reticulocyte lysate to translate Tetrahymena mRNAs into full-sized proteins . The activity of the subcellular fraction is shown to depend on the combined function of a protein component(s) and a tRNA(s) . The subcellular fraction is easily prepared and its usefulness for the identification of isolated mRNAs from Tetrahymena by their translation products in vitro is demonstrated.

J Cell Biol, 1987 Jun, 104(6), 1485 - 94
Tetrahymena contain two distinct and unusual high mobility group (HMG)-like proteins; Schulman IG et al.; Previous studies have described the existence of high mobility group (HMG)-like proteins in macronuclei of the ciliated protozoan, Tetrahymena thermophila (Hamana, K., and K . Iwai, 1979, J . Biochem . {Tokyo}, 69:1097-1111; Levy-Wilson, B., M . S . Denker, and E . Ito, 1983, Biochemistry, 22:1715-1721) . In this report, two of these proteins, LG-1 and LG-2, have been further characterized . Polyclonal antibodies raised against LG-1 and LG-2 fail to cross react with each other or any other macronuclear polypeptide in immunoblotting analyses . As well, LG-1 and LG-2 antibodies do not react with calf thymus, chicken, or yeast HMG proteins . Consistent with these results, a 47 amino-terminal sequence of LG-1 has been determined that shows limited homology to both calf thymus HMGs 1 and 2 and HMGs 14 and 17 . Two internal sequences of V8 protease-generated peptides from LG-2 have been determined, and these do not share any homology to the LG-1 sequence or any other sequenced HMG proteins . Comparison of the partial sequences of LG-1 and LG-2 with the complete amino acid sequence of the Tetrahymena histone H1 (Wu, M., C . D . Allis, R . Richman, R . G . Cook, and M . A . Gorovsky, 1986, Proc . Natl . Acad . Sci . USA, 83:8674-8678) rules out the possibility that LG-1 and LG-2 are proteolytically derived from H1, the other major macronuclear perchloric acid-soluble protein . Interestingly, however, both LG-1 and LG-2 are efficiently extracted from macronuclei by elutive intercalation (Schroter, H., G . Maier, H . Ponsting, and A . Nordheim, 1985, Embo (Eur . Mol . Biol . Organ.) J., 4:3867-3872), suggesting that both may share yet undetermined properties with HMGs 14 and 17 of higher eukaryotes . Examination of the pattern of LG-1 and LG-2 synthesis during the sexual phase of the life cycle, conjugation, demonstrates that the synthesis of LG-1 and LG-2 is coordinately increased from basal levels during the differentiation of new macronuclei (7-13 h), suggesting that both of these proteins play a role in determining a macronuclear phenotype . However, a specific induction of LG-2 synthesis is detected in early stages of conjugation (meiotic prophase, 1-4 h), leading to maximal synthesis of LG-2 at 3 h . Interestingly, the early induction of LG-2 synthesis closely parallels the hyperphosphorylation of histone H1 . Taken together, these data suggest that LG-1 and LG-2 are not strongly related to each other or to higher eukaryotic HMG proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

J Bacteriol, 1987 Jun, 169(6), 2380 - 4
2-Methylthio-1,4-naphthoquinone, a unique sulfur-containing quinone from a thermophilic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus; Ishii M et al.; A quinone was extracted and purified from the cells of an extremely thermophilic hydrogen bacterium, Hydrogenobacter thermophilus TK-6 (IAM 12695) . Its chemical structure was determined as 2-methylthio-3-VI, VII-tetrahydromultiprenyl-1,4-naphthoquinone by elemental analysis, 1H nuclear magnetic resonance spectroscopy, mass spectroscopy, and infrared spectroscopy of the quinone and by gas-liquid chromatography and gas chromatography-mass spectroscopy analysis of the ozonolysis products of the quinone . It was shown that the other five strains of H . thermophilus have the same quinone system . We named the quinone with the 2-methylthio-1,4-naphthoquinone nucleus "methionaquinone." The abbreviation of MTK is recommended for this class of quinone.

Biochem J, 1987 May 15, 244(1), 243 - 6
The interaction of spermine and pentamines with DNA; Basu HS et al.; We studied the effects of spermine, two naturally-occurring pentamines isolated from the thermophile Thermus thermophilus and one synthetic pentamine on the aggregation and 'melting' temperature of calf-thymus DNA and on the B-to-Z transition of poly(dG-me5dC) . All pentamines caused aggregation of DNA at much lower concentrations than that of spermine . Concentrations that increased the melting temperature of DNA and induced the B-to-Z transition in poly(dG-me5dC) were different for each pentamine, but were comparable with the concentration of spermine needed to cause these effects . Our results suggest that both the total charge and the distance separating the charge, which is a function of the length of the carbon chains between amino groups, are important for the induction of conformational changes in DNA . The biological role of pentamines in T . thermophilus appears to be related to their ability to promote DNA condensation at high temperature.

Eur J Biochem, 1987 May 15, 165(1), 147 - 55
Purification and characterization of D-glyceraldehyde-3-phosphate dehydrogenase from the thermophilic archaebacterium Methanothermus fervidus; Fabry S et al.; The D-glyceraldehyde-3-phosphate dehydrogenase from the extremely thermophilic archaebacterium Methanothermus fervidus was purified and crystallized . The enzyme is a homomeric tetramer (molecular mass of subunits 45 kDa) . Partial sequence analysis shows homology to the enzymes from eubacteria and from the cytoplasm of eukaryotes . Unlike these enzymes, the D-glyceraldehyde-3-phosphate dehydrogenase from Methanothermus fervidus reacts with both NAD+ and NADP+ and is not inhibited by pentalenolactone . The enzyme is intrinsically stable up to 75 degrees C . It is stabilized by the coenzyme NADP+ and at high ionic strength up to about 90 degrees C . Breaks in the Arrhenius and Van't Hoff plots indicate conformational changes of the enzyme at around 52 degrees C.

J Biol Chem, 1987 May 5, 262(13), 6334 - 8
Structural and functional relationship of ATP synthases (F1F0) from Escherichia coli and the thermophilic bacterium PS3; Steffens K et al.; Functional compatibility between the F1 and F0 parts of ATP synthases from Escherichia coli (EF1F0) and the thermophilic bacterium PS3 (TF1F0) was analyzed . F1-stripped everted membrane vesicles from both organisms bound the homologous or heterologous F1 part to the same extent . Titration of the reconstituted membrane vesicles with dicyclohexylcarbodiimide revealed a similar sensitivity of the homologous and hybrid F1F0 complexes towards the inhibitor . Furthermore, the heterologous enzymes exhibited ATP-dependent H+ translocation comparable to that of homologous F1F0 . Antisera raised against EF1 or subunits a, b, and c of EF0 were analyzed for cross-reactivity with TF1 and TF0 . Common antigenic sites have been detected with immunoblot analysis for subunit beta and subunit c of EF1F0 and the corresponding subunits from TF1F0 . A weak binding of the anti-a and anti-b antisera with the TF0 part has been observed in an enzyme-linked immunosorbent assay . Based on these findings the structural and functional relationship between the mesophilic and thermophilic ATP synthase complexes is discussed.

Mol Cell Biol, 1987 May, 7(5), 1725 - 30
Nonintegrative transformation in the filamentous fungus Podospora anserina: stabilization of a linear vector by the chromosomal ends of Tetrahymena thermophila; Perrot M et al.; The effect of the chromosomal ends of Tetrahymena thermophila on the stability of linear transforming molecules in the filamentous fungus Podospora anserina was tested . A derivative of an integrative vector for this fungus has been constructed, so that after linearization, the ends of the plasmid are the telomeric sequences of T . thermophila . After transformation, this linear molecule was maintained as an extrachromosomal plasmid with no integrated copies in about 50% of the transformants . Under selective conditions, there was approximately one linear molecule per 5 to 10 nuclei, and these extrachromosomal molecules were rapidly lost under nonselective conditions . The circular plasmid carrying an inverted repeat of T . thermophila telomeres could be linearized and processed in vivo.

Development, 1987 May, 100(1), 23 - 30
The assembly and positioning of cytoskeletal elements in Tetrahymena; Williams NE et al.; The oral skeleton of Tetrahymena is a precisely arranged assemblage of basal bodies, microtubule bundles and connecting filaments found associated with the feeding structure in this cell type . Tubulin and filament proteins have been isolated but no actin has been recovered . The conditional mutant NP1 of Tetrahymena thermophila forms a normal oral skeleton at the permissive temperature (28 degrees C), but forms an abnormal one at the restrictive temperature (37 degrees C) . Antibodies against tubulin and oral filament protein OF1 were used to visualize the abnormal oral skeleton and stages in its development, and ultrastructural comparisons of abnormal and wild-type oral skeletons were made . It was found that the overall pattern of organization was altered in the mutant, whereas the substructure appeared everywhere to be normal . Studies of cells in which the mutant phenotype was coming to expression revealed that normal basal bodies in the oral skeleton failed to move from the disordered state characteristic of early stages of development into the correct pattern of four organized clusters characteristic of later stages . Together, these results suggest that the lesion in NP1 does not affect cytoskeleton assembly per se, but instead affects a discrete mechanism responsible for the positioning of cytoskeletal elements with respect to each other after they have been formed (meta-assembly) . Reasons for suspecting the involvement of the membrane skeleton are presented.

Biochem J, 1987 May 1, 243(3), 779 - 87
Purification and properties of a stable beta-glucosidase from an extremely thermophilic anaerobic bacterium; Patchett ML et al.; A beta-glucosidase (EC 3.2.1.21) was purified to homogeneity from cell-free extracts of an extremely thermophilic anaerobic bacterium . The enzyme has an Mr of 43,000 as determined by molecular-exclusion chromatography, has a pI of 4.55 and shows optimum activity at pH 6.2 . The enzyme is active against a wide range of aryl beta-glycosides and beta-linked disaccharides, with beta-galactosidase activity only slightly less than beta-glucosidase activity, and significant beta-xylosidase activity . Lineweaver-Burk plots for p-nitrophenyl beta-glucoside, o-nitrophenyl beta-glucoside and cellobiose substrates are biphasic concave-downwards . Inhibition of the beta-glucosidase by substrates and glucose is negligible . Thermal inactivation follows first-order kinetics, with t1/2 (65 degrees C) 45 h, t1/2 (75 degrees C) 47 min and t1/2 (85 degrees C) 1.4 min and a deactivation energy of 380 kJ/mol at pH 6.2 . At pH 7.0, which is the optimum pH for thermostability, t1/2 (75 degrees C) is 130 min . At 75 degrees C, at pH 6.2, the thermostability is enhanced about 8-fold by 10% (w/v) glycerol, about 6-fold by 0.2 M-cellobiose and about 3-fold by 5 mM-dithiothreitol and 5 mM-2-mercaptoethanol.

J Dairy Sci, 1987 May, 70(5), 919 - 26
Enhancement of immune response in mice fed with Streptococcus thermophilus and Lactobacillus acidophilus; Perdigon G et al.; Swiss mice, fed for 8 consecutive d with 50 micrograms/d of viable cultures of Lactobacillus acidophilus and Streptococcus thermophilus, showed significant variation in their immune system . In order to study this phenomenon assays for macrophage and lymphocyte function were carried out . Both lactic acid bacteria enhanced significantly the enzymatic and phagocytic activity of peritoneal macrophages as checked against the controls and also accelerated the phagocytic function of the reticuloendothelial system as revealed by the carbon clearance test . On the 2nd d (100 micrograms), L . acidophilus reached a peak of K = .271, which remained high . Streptococcus thermophilus was effective only on the 2nd d and then decreased . The lymphocytic activity studied by immunoglobulin secreting cells was assayed by Jerne's method of plaque-forming cells (PFC) . This activity also was increased by the two microorganisms . Streptococcus thermophilus proved more effective than L . acidophilus . Lactobacillus acidophilus and S . thermophilus activated macrophages and lymphocytes and produced the same increase in the immune response of mice whether administered orally or intraperitoneally.

Biochim Biophys Acta, 1987 Apr 8, 912(2), 178 - 84
Amino-acid sequence of a tetrameric, manganese superoxide dismutase from Thermus thermophilus HB8; Sato S et al.; The amino-acid sequence of a tetrameric manganese superoxide dismutase from Thermus thermophilus HB8 has been determined . The protein was cleaved with cyanogen bromide (BrCN) into four peptides and their alignment was deduced through the fragment of partial cleavage with BrCN and the peptides were produced by cleavage of the protein with o-iodosobenzoic acid . Most of the peptides were sequenced by solid phase Edman degradation . Some of the peptides were sequenced by the Edman dansyl method after sub-fragmentation by proteinase digestion . The amino-acid sequence consists of 203 residues corresponding to a subunit molecular weight of 23,144.

Bioorg Khim, 1987 Apr, 13(4), 546 - 9
{Isolation and crystallization of phenylalanyl-tRNA-synthetase from Thermus thermophilus HB8}; Reshetnikova LS et al.; Method of isolation of phenylalanyl-tRNA synthetase from Thermus thermophilus HB8 is described, including chromatography on DEAE-sepharose, ammonium sulfate fractionation, hydrofobic chromatography on Toyopearl, gel filtration on ultrogel AcA-34, chromatography on phenylalanylaminohexyl-sepharose and heparine-sepharose . Yield of the purified enzyme was 10 mg from 1 kg of T . thermophilus cells . The enzyme is found to consist of two types of subunits with molecular masses 92 and 36 kDa and is likely to be a tetramer protein with molecular mass 250 kDa . Crystals of phenylalanyl-tRNA synthetase suitable for X-ray structural studies have been obtained.

J Gen Microbiol, 1987 Apr, 133 ( Pt 4), 1089 - 97
Molecular cloning and nucleotide sequence of the 3-isopropylmalate dehydrogenase gene of Candida utilis; Hamasawa K et al.; A 3-isopropylmalate dehydrogenase (3-IMDH, EC 1.1.1.85) gene was cloned from a gene library of Candida utilis . One of the plasmids, pYKL30, could complement Escherichia coli leuB and Saccharomyces cerevisiae leu2 auxotrophs; a 2.2 kb HindIII fragment subcloned in pBR322 could still complement the leuB mutation . Southern hybridization confirmed that this fragment was derived from C . utilis . An open reading frame of 1089 bp that corresponded to a polypeptide of 363 amino acids, one residue shorter than the 3-IMDH of S . cerevisiae, was found in the cloned fragment . The homology between the 3-IMDHs of C . utilis and S . cerevisiae was 76.2% in nucleotides and 85.4% in amino acids . In contrast, the homology between the 3-IMDHs of C . utilis and Thermus thermophilus was much smaller and was restricted to some regions of the gene.

Biol Chem Hoppe Seyler, 1987 Apr, 368(4), 353 - 67
Allophycocyanin complexes of the phycobilisome from Mastigocladus laminosus . Influence of the linker polypeptide L8.9C on the spectral properties of the phycobiliprotein subunits; Fuglistaller P et al.; The following phycobiliproteins and complexes of the allophycocyanin core were isolated from phycobilisomes of the thermophilic cyanobacterium Mastigocladus laminosus: alpha AP, beta AP, (alpha AP beta AP), (alpha AP beta AP)3, (alpha AP beta AP)3L8.9C, (alpha APB alpha AP2 beta AP3)L8.9C . The six proteins and complexes were characterised spectroscopically with respect to absorption, oscillator strength, extinction coefficient, fluorescence emission, relative quantum yield, fluorescence emission polarisation and fluorescence excitation polarisation . The interpretation of the spectral data was based on the three-dimensional structure model of (alpha PC beta PC)3 (Schirmer et al . (1985) J . Mol . Biol . 184, 257-277), which is related to the allophycocyanin trimer . The absorption and CD spectra of the complexes (alpha AP beta AP)3, (alpha AP beta AP)3L8.9C and (alpha APB alpha AP2 beta AP3)L8.9C could be deconvoluted into the spectra of the phycobiliprotein subunits . The assumptions made for the deconvolution could be checked by the synthesis of the spectra of (alpha APB beta AP)3 . The synthesised spectra are in good agreement with the corresponding measured spectra published by other authors . Considering the deconvoluted spectra the following influences on the chromophores could be ascribed to L8.9C: L8.9C neither influences the alpha AP nor the alpha APB chromophores . L8.9C shifts the absorption maximum of the beta AP chromophore to longer wavelength than the absorption maximum of the alpha AP chromophore in trimeric complexes . L8.9C increases the oszillator strength of the beta AP chromophores to about the value of the alpha AP chromophores in trimeric complexes . L8.9C turns the beta AP chromophores from sensitizing into weak fluorescing chromophores . By means of the hydropathy plot and the predicted secondary structure, a postulated three-fold symmetry in the tertiary structure of L8.9C could be confirmed.

J Dairy Sci, 1987 Apr, 70(4), 738 - 45
Purification and characterization of X-prolyl-dipeptidyl-aminopeptidase from Lactobacillus lactis and from Streptococcus thermophilus; Meyer J et al.; X-Prolyl-dipeptidyl-aminopeptidase recently was found in several lactic acid bacteria . This article describes the purification of the enzymes from Lactobacillus lactis and Streptococcus thermophilus and compares their characteristics . Enzymes from both strains are serine-peptidases . They both have a molecular weight of about 165,000 daltons, an isoelectric point near 4.5, and are constituted of two subunits . The pH optimum of the enzyme isolated from L . lactis is 7.0, whereas the enzyme from S . thermophilus possesses a broad pH optimum between 6.5 and 8.2 with glycyl-L-prolyl-aminomethylcoumarin as substrate . Below pH 5, both enzymes are unstable; however, that from S . thermophilus is more rapidly denatured . The enzyme from S . thermophilus is more sensitive to heat than the corresponding enzyme from L . lactis . Enzymes from the both strains have different specificities towards various substrates and are differently effected by metals, chelators, and other inhibitors . The importance of this enzyme for the metabolism of lactic acid bacteria is discussed.

J Mol Biol, 1987 Mar 20, 194(2), 181 - 92
Tetrahymena actin . Cloning and sequencing of the Tetrahymena actin gene and identification of its gene product; Hirono M et al.; Actin is ubiquitous in eukaryotes, nevertheless its existence has not yet been clearly proven in Tetrahymena . Here we report the cloning and sequencing of an actin gene from the genomic library of Tetrahymena pyriformis using a Dictyostelium actin gene as a probe . The Tetrahymena actin gene has no intron . The predicted actin is composed of 375 amino acids like other actins and its molecular weight is estimated as 41,906 . Both T . pyriformis and T . thermophila possess a single species of actin genes which differ in their restriction patterns . Northern hybridization analysis revealed that the actin gene was actively transcribed in vivo . To detect the gene product, we synthesized an N-terminal peptide of the deduced sequence and prepared its antibody . Using an immunoblotting technique, we identified Tetrahymena actin on a two-dimensional gel electrophoretic plate . The actin spot migrated near an added spot of rabbit skeletal muscle actin, but clearly differed from the latter in its isoelectric point and apparent molecular weight . The primary structure of Tetrahymena actin shares about 75% homology equally with those of other representative actins . This value is extremely low as a homology rate between known actins . Tetrahymena actin diverges not only in relatively variable regions of other actins, but also in relatively constant regions . The hydrophilicity levels of two regions (residues 190 to 200 and residues 225 to 235) are also quite different between the Tetrahymena actin and skeletal muscle actin . Thus, we conclude that actin is present in Tetrahymena, but it is one of the most unique actins among the actins known hereto.

Nucleic Acids Res, 1987 Mar 11, 15(5), 1905 - 20
Characterization of an authentic intermediate in the self-splicing process of ribosomal precursor RNA in macronuclei of Tetrahymena thermophila; Kister KP et al.; We have characterized a 1.5 kb RNA species in T . thermophila macronuclei previously found in vivo and including intron sequences linked to the 3' exon . This IVS-3' exon RNA could be detected in gels as a discrete molecule only after denaturation of nuclear RNA . After addition of 32P-GTP, as splicing cofactor in a nuclear in vitro system, the IVS-3' exon RNA was labeled at its 5' terminus, as was the by-product of splicing, the excised IVS RNA . The time course of labeling indicates that the IVS-3' exon RNA acts like a reaction intermediate and specifically a kinetic precursor to IVS RNA . Partial nuclease digestions showed that the IVS-3' exon RNA and the IVS RNA have the same 5' terminal sequence . In addition the IVS-3' exon RNA can release the 15-mer oligonucleotide cleaved off during circularization of IVS RNA under conditions of high temperature . Taken together, the structural, functional, and kinetic properties of the IVS-3' exon RNA strongly suggest that it represents a previously postulated in vivo intermediate in the splicing pathway.

J Bacteriol, 1987 Mar, 169(3), 1328 - 30
Effect of growth temperature on the long-chain diols and fatty acids of Thermomicrobium roseum; Pond JL et al.; Long-chain 1,2-diols constitute the hydrophobic backbone of membrane lipids (replacing glycerolipids) in the thermophilic eubacterium Thermomicrobium roseum . The effects of incubation temperature on chain length and chain branching of diols and fatty acids were investigated . The percentage of branched chains decreased, and chain length increased slightly, with increased growth temperatures.

Exp Cell Res, 1987 Mar, 169(1), 63 - 73
Unidirectional co-stimulation by a non-mating strain of Tetrahymena thermophila; Katz M et al.; We report that a fatty acid auxotroph of Tetrahymena thermophila (RH179E1) fails to mate, yet retains the ability to co-stimulate normal cells unidirectionally . Thus, co-stimulation can be analyzed experimentally in the absence of pair formation . We show that the co-stimulation of normal cells of one mating type is sufficient to shorten the waiting period for pair formation of those cells with initiated cells . This is the first evidence that co-stimulation causes a hyperinduction of mating reactivity in T . thermophila, generating in turn a positive feedback mechanism for (presumably) gamone production . Co-stimulation by the variant strain is at a maximum after 3-4 h of exposure when the variant and wild-type cells are at a ratio of 1:1 . When mixed with wild-type cells, RH179E1 induces the formation of progeny (at low frequency) which inherit exclusively genetic material of the wild-type cells.

J Appl Bacteriol, 1987 Mar, 62(3), 209 - 16
Conventional taxonomy of lactobacilli surviving radurization of meat; Hastings JW et al.; All of the 113 catalase-negative, Gram-positive, rod-shaped strains isolated from radurized minced beef (5 kGy) were homofermentative, non-thermophilic, and belonged to the sub-genus Streptobacterium . The majority of the strains (100) were identified as Lactobacillus sake . These were divided into four sub-groups based on their sugar fermentation pattern: group IA1 (melibiose (+), maltose (-), amygdalin (-), 76 strains); group IA2 (melibiose (+), maltose (-), amygdalin (+), 14 strains); group IB1 (melibiose (+), maltose (+), amygdalin (+), four strains); group IB2 (melibiose (+), maltose (+), amygdalin (-), six strains) . Of the remaining strains, two produced L(+)-lactic acid and were identified as L . farciminis, three were identified as L . curvatus and eight showed characteristics of both L . sake and L . curvatus and were designated 'L . sake/curvatus.' With one exception, all strains were aciduric and relatively insensitive to the chemical preservatives tested . Most L . sake strains produced significant amounts of H2O2 . Electron microscopy confirmed a possible relationship between the thickness of the cells and radiation resistance . The problems and limitations of this type of taxonomic study and possible reasons for the predominance of L . sake species in radurized meat are discussed.

J Dairy Sci, 1987 Mar, 70(3), 514 - 23
Oxygen uptake activity and aerobic metabolism of Streptococcus thermophilus STH450; Teraguchi S et al.; Streptococcus thermophilus STH450 had a very high oxygen uptake . This strain was then compared with aerobic metabolism to S . thermophilus ATCC 19258, a reference strain for aerobic metabolism . Molecular oxygen, which was absorbed by S . thermophilus STH450 during aerobic glycolytic metabolism, was involved in the oxidation of NADH by the catalytic activity of NADH oxidase . The portion of pyruvate that corresponded to the oxidized NADH was committed to form alpha-acetolactate, acetoin, and diacetyl . Both strains were deficient in peroxidase and pyruvate oxidase activities; therefore, NADH oxidase was probably the terminal oxidase in aerobic glycolytic metabolism . Oxygen uptake and NADH oxidase activities were significantly higher in S . thermophilus STH450 than in S . thermophilus ATCC 19258 . alpha-Acetolactate, acetoin, and diacetyl also accumulated during aerobic glycolytic metabolism of S . thermophilus STH450 . However, when both strains were grown in the presence of pyruvate, these metabolites were equivalent . Hence, less oxygen might be needed for pyruvate metabolism.

J Biochem (Tokyo), 1987 Mar, 101(3), 633 - 42
Evidence for two activated forms of pyruvate kinase from Bacillus stearothermophilus in the presence of ribose 5-phosphate; Sakai H et al.; Allosteric activation of pyruvate kinase from a thermophilic bacterium, Bacillus stearothermophilus, by ribose 5-phosphate (R5P) was kinetically examined . Two activated forms of this enzyme could be distinguished, depending on the R5P concentration . One form (Form I) was observed at about 10(-5) M R5P . It showed a slightly negative cooperativity for phosphoenolpyruvate (PEP) . The other form (Form II) was observed at more than 10(-3) M R5P and showed Michaelis-Menten kinetics for PEP . The PEP and ADP concentrations that yield half-maximal velocity were essentially identical for the two forms (about 0.1 and about 0.5 mM, respectively), but Form I had a larger Vmax value than Form II . In the absence of R5P, the enzyme showed a homotropic positive cooperativity for PEP; the concentration required for the half-maximal velocity was about 2 mM and that of ADP was about 1.6 mM . The enzyme was more susceptible to protease digestion in the presence of R5P than in the absence of it . The concentration of R5P required for the enzyme to be susceptible to protease digestion was approximately identical with that required to generate Form I . With more than 10(-3) M R5P, the thermostability of the enzyme was greatly increased . The concentration of R5P required for the enzyme to be thermostable was in good agreement with that required to generate Form II . These results indicate that the two activated forms distinguished kinetically differ in their conformations, too . The saturating level of PEP did not cause such a change in the thermostability or the susceptibility to protease.

Biochim Biophys Acta, 1987 Feb 27, 908(2), 150 - 7
Purification and characterization of an inducible mitochondrial DNA polymerase from Tetrahymena thermophila; Ostergaard E et al.; Treatment of the eukaryotic organism Tetrahymena with various types of DNA-damaging agents has been reported to cause a 35-fold induction of a mitochondrial DNA polymerase . We here report that the enzyme can be induced in large-scale cultures by exposure of the cells to thymine starvation and/or intercalating agents . The induced DNA polymerase has been purified to near homogeneity, with a specific activity of approx . 300,000 units/mg protein . The relative molecular mass of the active form of the enzyme is approx . 100,000, as determined by glycerol gradient sedimentation . The subunit structure has been analysed by SDS polyacrylamide gel electrophoresis of the highly purified preparation and by immunoprecipitation with a monoclonal antibody directed to the DNA polymerase . A polypeptide of Mr 47,000 has been observed to be a subunit of the enzyme . This corresponds to the size of the subunits suggested for mitochondrial DNA polymerase from chicken embryos and mouse myeloma cells.

Nucleic Acids Res, 1987 Feb 11, 15(3), 921 - 32
Syntheses of rRNA, 5.8S, 5S and tRNA are inhibited equally by 8-methoxypsoralen phototreatment of Tetrahymena thermophila; Nielsen PE; Treatment of the ciliated protozoan Tetrahymena thermophila with 8-methoxypsoralen combined with long wavelength ultraviolet irradiation (UVA, lambda approximately 360 nm) resulted in a dose dependent equal inhibition of the synthesis of rRNA, 5.8S, 5S and tRNA . Similar results were obtained with 3-carbethoxy-8-methoxypsoralen which predominantly forms DNA mono-adducts . In contrast the synthesis of tRNA in T . thermophila was much less sensitive than that of rRNA, 5.8S and 5S RNA to treatment with short wavelength ultraviolet irradiation (UVB, lambda approximately 254 nm) . These results are interpreted in favor of a mechanism by which psoralen-DNA adducts (crosslinks much greater than monoadducts) inhibit RNA transcription initiation (in contrast to UVB which causes premature chain termination) . Furthermore it is argued that RNA synthesis is regulated in equally sized domains regardless of the gene-size.

J Appl Toxicol, 1987 Feb, 7(1), 35 - 41
Discovery of multiple organofluorophosphate hydrolyzing activities in the protozoan Tetrahymena thermophila; Landis WG et al.; Recently it has been found that homogenates of Tetrahymena thermophila can hydrolyze the potent acetylcholinesterase inhibitors O,O-diisopropylphosphofluoridate (DFP) and O-1,2,2-trimethylpropylmethylphosphonofluoridate (soman) . Upon purification of the DFP hydrolyzing activity 10-fold it had been noted that the soman hydrolyzing activity increased only 2-3 fold . Treatment with manganous ion and comparison of the soman and DFP hydrolysis rates of the homogenate indicated that a mixture of the squid-type and Mazur-type DFPases may be present . Subsequent purification of the enzymatic activities within the Tetrahymena-homogenate demonstrated that there are at least five functioning proteins of molecular weights 67,000 to 96,000 . None are directly homologous to the DFPases found in hog kidney or squid . The enzymatic activities are designated DFPase-1 through DFPase-5 . A hypothesis is presented that the functions of DFPases are in the normal metabolism of organophosphates naturally synthesized by T . thermophila.

J Appl Bacteriol, 1987 Feb, 62(2), 167 - 76
A study of thermophilic campylobacters in a river system; Bolton FJ et al.; Fifteen kilometres of a river system traversing rural and urban areas and subject to sewage works effluent discharge was studied during a 12 1/2 month period . A total of 312 samples was collected from 12 sites at 14 d intervals and tested by a glass microfibre filtration method and a most probable number (MPN) method . Campylobacters were found in 43% of samples by the filtration method and 21% by the MPN method . The lowest frequency of isolation and lowest counts (less than 10 campylobacters/100 ml) were associated with samples collected from rural sites and fast-flowing stretches of river . The greatest frequency of isolation and highest counts (greater than 10-230 campylobacters/100 ml) were associated with sites adjacent to or downstream of sewage works . There was an obvious seasonal trend; most isolations and highest counts were obtained in late autumn and winter, and fewest isolations and lowest counts in spring and summer . Surface water run-off from adjacent farmland following heavy rainfall also increased the counts of campylobacters in the river system . Biotyping of isolates demonstrated that the most prevalent Campylobacter sp . was Campylobacter jejuni but C . coli, C . laridis and a previously unrecognized group of campylobacters were also isolated . Serotyping differentiated 14 serotypes of C . jejuni, 11 of C . coli and two of C . laridis . Furthermore, serotypes of C . jejuni commonly isolated from enteritis in man were frequently found in river water tested during this study.

Biol Chem Hoppe Seyler, 1987 Feb, 368(2), 121 - 30
Ribosomal proteins and DNA-binding protein II from the extreme thermophile Bacillus caldolyticus; Beck A et al.; Crystallographic studies, presently on ribosomal and DNA-binding proteins from the moderate thermophile Bacillus stearothermophilus, can be expected to benefit from the use of even more stable proteins from extreme thermophiles . Bacillus caldolyticus, which is able to grow in the temperature range of 70-80 degrees C, appears to be a suitable candidate . We have compared the two bacilli using two criteria: the two-dimensional gel patterns of ribosomal proteins and the properties of DNA-binding protein II . The latter protein is ubiquitous in the eubacterial kingdom and can be purified in large quantities . B . caldolyticus can be grown at 75 degrees C in continuous culture with a generation time of 45-60 min . The yield of ribosomes compares favorably with that of B . stearothermophilus . The gel patterns of the ribosomal proteins are very similar but several differences, in particular among the 50S proteins, are observed . The N-terminal amino-acid sequence of the DNA-binding protein differs in 3 positions (out of 39) from B . stearothermophilus and the protein shows an increased resistance to thermal denaturation . Tetragonal and monoclinic crystals of DNA-binding protein II have been obtained which are suitable for X-ray studies and the diffraction patterns of the two crystal forms are shown.

Proc Natl Acad Sci U S A, 1987 Feb, 84(3), 675 - 9
Control of oligomeric enzyme thermostability by protein engineering; Ahern TJ et al.; The ability to control the resistance of an enzyme to inactivation due to exposure to elevated temperatures is essential for the understanding of thermophilic behavior and for developing rational approaches to enzyme stabilization . By means of site-directed mutagenesis, point mutations have been engineered in the dimeric enzyme yeast triosephosphate isomerase that improve its thermostability . Cumulative replacement of asparagine residues at the subunit interface by residues resistant to heat-induced deterioration and approximating the geometry of asparagine (Asn-14----Thr-14 and Asn-78----Ile-78) nearly doubled the half-life of the enzyme at 100 degrees C, pH 6 . Moreover, in an attempt to model the deleterious effects of deamidation, we show that replacement of interfacial Asn-78 by an aspartic acid residue increases the rate constant of irreversible thermal inactivation, drastically decreases the reversible transition temperature, and reduces the stability against dilution-induced dissociation.

Nucleic Acids Res, 1987 Jan 26, 15(2), 683 - 93
Structure determination of a new fluorescent tricyclic nucleoside from archaebacterial tRNA; McCloskey JA et al.; A highly fluorescent nucleoside was detected in enzymatic digests of the extremely thermophilic archaebacterium Sulfolobus solfataricus by combined liquid chromatography-mass spectrometry (LC/MS) . Following isolation, the structure was determined primarily by mass spectrometry, to be 3-(beta-D-ribofuranosyl)-4,9-dihydro-4,6,7-trimethyl-9-oxoimidazo{ 1, 2-a}purine (mimG), a new derivative of the Y (wye) nucleoside . The structural assignment was verified by comparison of the base released by acid hydrolysis with the corresponding synthetic base, using mass spectrometry, chromatography, and UV absorption and fluorescence properties . Nucleoside mimG was also detected by LC/MS in hydrolysates of the thermophiles Thermoproteus neutrophilus and Pyrodictium occultum . These results constitute the first finding of a member of the hypermodified Y family of nucleosides in archaebacteria.

J Biol Chem, 1987 Jan 15, 262(2), 558 - 63
Functions of isolated domains of methionyl-tRNA synthetase from an extreme thermophile, Thermus thermophilus HB8; Kohda D et al.; Methionyl-tRNA synthetase (MetRS, 2 X 75 kDa) was purified to homogeneity from an extreme thermophile, Thermus thermophilus HB8 . The polypeptide chain of MetRS was cleaved by limited digestion with trypsin into four domains: T1 (29 kDa), T2 (23 kDa), T3 (14.5 kDa), and T4 (7.5 kDa), which were aligned in that order . MetRS was also cleaved into similar fragments with a variety of other proteases . Domains T1, T2, T3, and T4 were isolated by column chromatography . "Tandem domain" T1-T2 (56 kDa) is fully active in the aminoacylation of tRNA and is further cleaved with trypsin into domains T1 and T2 . Domain T1 is the smallest aminoacylation unit so far reported . Domain T2 (enzymatically inactive) interacts with tRNAMetf, as found by UV-induced cross-linking . Isolated domain T3 forms a dimer and is responsible for the dimer assembly of two protomers in MetRS . Domain T4 is a flexible tail of MetRS . These domains, in particular T1 and T2, will be important for detailed structure analyses in relation to aminoacylation activity.

Nucleic Acids Res, 1987 Jan 12, 15(1), 141 - 60
Unusual features of transcribed and translated regions of the histone H4 gene family of Tetrahymena thermophila; Horowitz S et al.; The complete DNA sequence is presented of H4-II, the second of the pair of histone H4 genes of the ciliated protozoan, Tetrahymena thermophila . Both H4 genes code for the same protein . Codon usage in these and other Tetrahymena genes is severely restricted and is similar to that in yeast . Flanking regions are AT-rich (greater than or equal to 75%), relative to coding sequences (approximately 45% GC) . Except for small, similarly positioned homologies, flanking sequences of the two genes are different . Canonical sequences in higher eukaryotic promoters are not obvious in these genes . Instead, short, localized, base composition eccentricities characterize the 5' flanking sequences of all Tetrahymena genes analyzed . The consensus, P yP u(A)3-4 ATGG initiates translation in these and all other known Tetrahymena genes . Nuclear transcripts and messages of both growing and starved cells begin at multiple sites, mainly at the first or second A residue following a pyrimidine . The palindrome typical of histone message 3' termini in higher organisms is not present . Downstream of both genes are sequences similar to the processing/polyadenylation signal of higher eukaryotes, although the unique 3' ends are not those predicted by the location of the signals.

Biochim Biophys Acta, 1987 Jan 5, 911(1), 81 - 94
Spectroscopic studies of the seven-iron-containing ferredoxins from Azotobacter vinelandii and Thermus thermophilus; Johnson MK et al.; The seven-iron-containing ferredoxins from Azotobacter vinelandii and Thermus thermophilus have been investigated by low-temperature magnetic circular dichroism (MCD) and electron paramagnetic resonance (EPR) spectroscopies and room temperature ultraviolet-visible absorption spectroscopy . The results confirm the presence of one trinuclear and one tetranuclear iron-sulfur cluster in both ferredoxins and facilitate comparison of the electronic and magnetic properties of the oxidized and reduced {3Fe-xS} clusters . MCD magnetization data are consistent with an S = 2 ground state for both reduced {3Fe-xS} clusters, but indicate differences in the rhombicity of the zero-field splittings . The data permit rationalization of the absence of a delta M = 4 EPR transition for the reduced {3Fe-xS} cluster in A . vinelandii ferredoxin I . Spectroscopic studies of anaerobically isolated A . vinelandii ferredoxin I do not support the hypothesis that the {3Fe-xS} cluster arises as a result of aerial oxidative damage to a {4Fe-4S} cluster during isolation . The possibility that two distinct forms of {3Fe-xS} clusters can exist in A . vinelandii ferredoxin I was investigated by spectroscopic studies as a function of pH . The results reveal two distinct and interconvertible forms of the reduced {3Fe-xS} cluster, but do not permit rationalization of the inconsistencies in the structural data that have been reported for the oxidized clusters.

Nucleic Acids Symp Ser, 1987, (18), 277 - 80
Self-splicing of Tetrahymena rRNA can proceed with phosphorothioate substitution at the splice sites; Deeney CM et al.; The self-excision of a 413-base intervening sequence of the 26S rRNA of Tetrahymena thermophila has been investigated using phosphorothioate-substituted RNA . Transcripts containing this intron were prepared by T7 RNA polymerase-catalyzed polymerisation using a M13 mICE10 vector in the presence of various nucleoside alpha-thiotriphosphate analogues . Wild-type transcripts incorporating phosphorothioates 5' to adenosine or uridine were inactive, whereas incorporation 5' to cytidine or guanosine allowed splicing . The first two substitutions place phosphorothioates inter alia at the 5' and 3' splice sites respectively . Mutagenesis at either site allowed phosphorothioate substitution 5' to guanosine at each splice site . This did not block splicing, suggesting that substitution at internal sites within the intron has more effect.

Folia Microbiol (Praha), 1987, 32(4), 354 - 9
Bioconversion and binding of sterols by thermophilic moulds; Satyanarayana T et al.; None of the fourteen thermophilic moulds was able to break down the aliphatic side chain of sterols, viz . cholesterol, lanosterol, sitosterol, and stigmasterol so as to yield 4-androstene-3,17-dione, 1,4-androstadiene-3,17-dione and progesterone . In Acremonium alabamensis and Talaromyces emersonii, cholestenone was detected as a product of fermentation of cholesterol whereas the former yielded stigmastadienone from stigmasterol and sitosterol . Lanosterol appeared to be resistant to fungal bioconversion . All the thermophilic moulds exhibited avidity for binding sterols to the mycelium, but the ability to bind sterol seemed to depend upon the nature of the organism and the sterol.

Antonie Van Leeuwenhoek, 1987, 53(2), 85 - 91
Lipid composition of thermophilic moulds Acremonium alabamensis and Thermomucor indicae-seudaticae; Satyanarayana T et al.; The total lipid content of Acremonium alabamensis and Thermomucor indicae-seudaticae ranged 2.6-7.3 and 8.5-13.0% of dry mycelium, respectively during development . Neutral lipid fraction increased during growth while polar and phospholipids declined . Both moulds contained palmitic, oleic, linoleic and palmitoleic acids as major fatty acid components in lipids . Degree of unsaturation of lipids of A . alabamensis was greater than that of T . indicae-seudaticae . Neutral lipids were more unsaturated than the polar lipids . The ratio of unsaturation index of polar lipids to neutral lipids was either one or less than one . The principal phospholipids of these moulds were phosphatidyl choline, phosphatidyl ethanolamine and phosphatidic acid . However, phosphatidic acid was not found in very high amounts as observed in Humicola grisea var . thermoidea.

Development, 1987 Jan, 99(1), 51 - 68
Positional reorganization in compound janus cells of Tetrahymena thermophila; Frankel J et al.; The janus mutations of Tetrahymena thermophila convert the large-scale organization of the dorsal surface of the cell into a mirror-image of the ventral surface, which is characterized by a second, abnormal, oral apparatus and by contractile vacuole pores to the left of the second oral area rather than the usual right . This conversion could be due either to a local change in the response to an unaltered positional system or to a more global reorganization of the system itself . janus homopolar doublets were used to distinguish between these two alternatives . Homopolar doublets can be made by fusing two similarly oriented cells in side-by-side parabiosis . Non-janus homopolar doublets typically possess two sets of normal oral structures with contractile vacuole pores to the right of each of them . In janus doublets, there are up to four sets of oral structures, with the abnormal oral structures located between the two sets of normal oral structures; contractile vacuole pores are situated to the right of the normal oral areas and to the left of the abnormal oral structures . Non-janus homopolar doublets are known to propagate their compound condition for a number of cell divisions, but also to regulate toward the singlet state through a progressive reduction in number of ciliary rows followed by loss of one of the two sets of major cell surface structures . janus homopolar doublets go through a corresponding regulation . As a consequence, the location of the abnormal oral structures relative to the normal ones is more variable in janus doublets than in janus singlets . Sometimes the abnormal oral structures shift to a position close to their normal counterparts and then the intervening CVP sets disappear . There is evidence for occasional fusion of an abnormal oral area with an adjacent normal oral apparatus, a condition that may be transitional to the singlet state . These observations are inconsistent with the idea of a fixed positional system and strongly suggest a global reorganization of the surface pattern in a manner consistent with predictions of an intercalation model that was first proposed to explain the regulation of non-janus doublets to singlets.

Thorax, 1987 Jan, 42(1), 32 - 7
Extrinsic allergic alveolitis caused by a cold water humidifier; Robertson AS et al.; Three workers developed classical extrinsic allergic alveolitis while working in a printing works that had a contaminated cold water humidifier . All had nodular shadows on their chest radiographs, reduced gas transfer measurements, and lung biopsy specimens that showed an alveolitis with giant cells and cholesterol clefts . In two subjects bronchoalveolar lavage was performed and the lavage fluid contained more than 70% lymphocytes in each case . Bronchial provocation tests with the humidifier antigen in these two workers reproduced their symptoms . Unlike previously reported cases, where exposure was to humidifiers working at generally higher temperatures, challenge with thermophilic actinomycetes in our two patients produced no reaction . Tests for precipitins to the humidifier antigen gave strongly positive reactions in the three workers but no single organism isolated from the humidifier produced a significantly positive reaction.

Int J Biochem, 1987, 19(6), 545 - 50
Ornithine decarboxylase from Tetrahymena thermophila: purification, reversible modification and multiple forms; Yao KM et al.; T . thermophila ornithine decarboxylase was purified 1100-fold by an improved procedure to a specific activity of 2.31 x 10(4) nmol/hr/mg protein with a 28% yield . The purified enzyme was a monomer of Mr = 68,000 and had two isoelectric forms with pl's of 5.14 and 5.17 respectively . The Mr = 68,000 enzyme was reversibly modified by a cellular factor of approx . Mr = 10,000 to give rise to two additional forms with decreased DEAE-cellulose affinity.

FEBS Lett, 1987 Jan 1, 210(1), 85 - 90
The complete amino acid sequences of the 5 S rRNA binding proteins L5 and L18 from the moderate thermophile Bacillus stearothermophilus ribosome; Kimura J et al.; The complete amino acid sequences of the 5 S rRNA binding proteins L5 and L18 isolated from ribosomes of the moderate thermophile Bacillus stearothermophilus are presented . This has been achieved by the sequence analysis of peptides derived by enzymatic digestions with trypsin, chymotrypsin, pepsin, and Staphylococcus aureus protease, as well as by chemical cleavage with cyanogen bromide . The proteins L5 and L18 consist of 179 and 120 amino acid residues, and have Mr values of 20,163 and 13,473, respectively . A comparison of the sequences with their counterparts from the Escherichia coli ribosome reveals 59% identical residues for L5, and 53% for L18 . For both proteins, the distribution of conserved regions is not random along the protein chains: some regions are highly conserved while others are not . The regions which are conserved during evolution may be important for the interaction with the 5 S rRNA molecule.

Eur J Respir Dis Suppl, 1987, 152, 91 - 100
Airborne moulds and actinomycetes in work environment of farmers; Kotimaa MH et al.; The aim of this series of studies was to investigate the quality and quantity of farmers' exposure to airborne spores during the handling of hay or grain . In the beginning, the Petri dish method and later a six-stage Andersen sampler were used to collect the samples . The number of spores of mesophilic fungi, thermotolerant fungi, thermophilic actinomycetes and fungi of the Aspergillus glaucus group were determined in order to find possible causative agents of farmer's lung disease . The level of exposure varied from 10(4) cfu/m3 to 10(7) cfu/m3 (cfu = colony forming unit) . In hay, fungi of the A . glaucus group usually dominated . In grain the most common moulds were Cladosporium spp . and Penicillium spp . In both hay and grain the most common thermophilic actinomycete was Thermoactinomyces vulgaris; Micropolyspora faeni was found less frequently . Silaging was found to be the best method to prevent moulding of hay . Chemicals added during baling did not satisfactorily prevent moulding of hay . For stored grain, however, the best results were obtained with propionic acid treatment . The quality and quantity of airborne spores found suggests that farm work exposes farmers to a high risk of becoming sensitized, which leads to the development of asthma or farmer's lung . Few of the methods presently available for making or storing hay and grain can satisfactorily prevent moulding . So far, use of personal dust respirators with a type P2 (previously II b) filter seems to be the only way to effectively diminish exposure to spores.

Proc Natl Acad Sci U S A, 1987 Jan, 84(2), 383 - 7
A heat shock-induced, polymerase III-transcribed RNA selectively associates with polysomal ribosomes in Tetrahymena thermophila; Kraus KW et al.; Tetrahymena thermophila cells, subjected to a heat shock-inducing temperature, manifest a number of translationally regulated changes during the course of a continuous heat shock treatment . One particular change, the resumption of translation of mRNAs coding for normal cellular proteins, was found to correlate with a polysomal ribosome association not found prior to heat shock . A low molecular weight RNA (ca . 270 nucleotides), whose rapid accumulation was induced by heat shock, became quantitatively associated with polysomal ribosomes during that time when normal cell protein synthesis became reestablished . We estimated that there were one or two of these RNAs per ribosome uniformly distributed throughout the polysomal ribosome population . The gene (or genes) coding for this RNA were found to be transcribed by polymerase III.

Boll Ist Sieroter Milan, 1987, 66(6), 456 - 65
{Bacteriostatic and bactericidal activity, resistance and tolerance of 16 strains of thermophilic Campylobacter to 12 antibiotic drugs}; Moretti MV et al.; The majority of gastrointestinal infections due to "thermophilic" Campylobacter is self limiting and does not need antibiotic treatment . Anyhow there are some serious cases (sepsis, persistent and relapsing gastroenteritis, severe immunodeficient patients) which require appropriate therapy . The susceptibility of 15 strains of Campylobacter jejuni and of 1 strain of C . coli, isolated from patients with acute gastroenteritis, has been studied against 12 antibiotics with the broth microdilution method at two different inocula (10(3)-10(4) CFU/ml and 10(7)-10(8) CFU/ml), and with the standard agar disk diffusion test, modified to allow sensitivity testing of Campylobacter . For each antibiotic, the geometric mean of MIC and of MBC and the concentrations of the various drugs needed for inhibition and killing of 50 and 90% of the strains (MIC-MBC50 and MIC-MBC90 respectively) have been calculated . Finally the percentage of resistant strains and the percentage of tolerant strains (ratio MBC/MIC: greater than or equal to 32) at low and high inoculum was determined . Erythromycin and aminoglycosides resulted the most active antibiotics against Campylobacter, being bactericidal as well as bacteriostatic at both low and high inoculum . Among the beta-lactams, cefotaxime was the most active, followed by piperacillin and ampicillin . Ceftazidime, aztreonam and rifampin were inactive . Ciprofloxacin, cotrimoxazole and tetracyclines showed some activity against Campylobacter at low inoculum . The agar disk diffusion method cannot be used for the "routinary" assay of susceptibility of Campylobacter, because it is a "naggy" microaerophilic organism.

Symp Soc Exp Biol, 1987, 41, 21 - 33
Temperature and macromolecular structure and function; Pain RH; Stability is frequently a knife-edge phenomenon and it is this aspect which is both essential for the effective involvement of macromolecules in the living cell and also provides the basis for the sensitivity of some macromolecular systems to temperature . The response of proteins and nucleic acids is relatively simple at the phenomenological level, with rather sharp 'melting' transitions occurring . Since however the thermodynamic stability of both depends on the cooperation of a variety of non-covalent interactions which are qualitatively well understood but quantitatively difficult to assess, the full understanding and prediction of structural stability still evades us . We shall consider the effects of temperature on the different non-covalent interactions and how far these can account for protein and nucleic acid denaturation at elevated temperatures and also the cold inactivation of proteins . The latter has recently been shown to involve unsuspected complications in terms of protein conformational change . The increased stability of proteins and nucleic acids from thermophiles will be discussed . Stability is important not only in native folded proteins but also with respect to intermediate structures which occur during the folding of the newly synthesized polypeptide chain into the native, active protein . Through studies of protein folding the molecular basis of the phenomenon of temperature sensitive synthesis has been revealed . Given a stable molecular structure, its function will frequently be subject to variation with temperature . The deceptively simple temperature dependence of enzyme activity will involve the non-covalent interactions considered above for interaction between enzyme and substrate and for stability of the transition state complex . This complexity again makes the temperature dependence difficult to interpret . Further, the fact that proteins are dynamic structures is becoming recognized as an important feature factor in determining function . A balance has to be struck between on the one hand dynamic mobility which is essential for catalytic activity and on the other thermodynamic stability which holds the molecule in a potentially functional conformation under the given conditions of temperature and pressure . Readjustment of thermostability stability, as in thermophiles (or vice versa?), must also involve readjustment of dynamic mobility.(ABSTRACT TRUNCATED AT 250 WORDS)

Ann N Y Acad Sci, 1987, 506, 371 - 83
Some factors affecting the copy number of specific plasmids in Bacillus species; Imanaka T; Some factors affecting the copy number of specific plasmids in different hosts were presented . A low copy number (one copy per chromosome) Kmr Tcr plasmid pTB19 was isolated from thermophilic bacillus . We have constructed 22 derivatives from pTB19 and their copy numbers range from 1 to 214 per chromosome in B . subtilis and B . stearothermophilus . Some recombinant plasmids containing the specific 1.0-MDa EcoRI fragment exhibited high transformation frequency and low copy number in B . stearothermophilus and were stable in the thermophile; on the other hand, those plasmids were unstable in different host B . subtilis, as mentioned earlier . By selecting the best combination of vector plasmid and host strain, both molecular cloning of various enzyme genes (i.e., penicillinase, thermostable alpha-amylase, and thermostable neutral protease) and enhancement of the enzyme production could be easily achieved.

Int J Biochem, 1987, 19(5), 483 - 6
Some observations on the inhibition and activation of a thermophilic protease; Cowan DA et al.; The activity of Caldolysin, the extracellular protease from the extreme thermophile Thermus aquaticus strain T351, was reduced in the presence of high protein concentrations . The absence of this effect after enzyme immobilization, or when using chromogenic substrates, suggests that a steric mechanism is involved . The apparent activation of caldolysin under certain conditions was shown to be related to both temperature and the ionic strength of the aqueous environment . The effects on activity, substrate affinity and thermostability of chemical modification with various reagents are also discussed.

Histochemistry, 1987, 87(6), 619 - 22
Cytochemical investigation into the Ca-dependence of positive and negative hormonal imprinting in Tetrahymena; Kovacs P et al.; Insulin gives rise to positive imprinting in Tetrahymena pyriformis, but to negative imprinting in T . thermophila, as revealed by the respective increases and decreases in the insulin-binding capacity of these organisms observed during later interactions with this hormone . We found that changes in insulin-binding capacity exhibited parallelism with fluctuations of the levels of free, intracellular Ca2+ detectable by Quin-2 labeling . An exception was the second interaction of T . thermophila with insulin, which although showing a positive trend, produced a relatively small increase in the level of intracellular Ca2+ . These observations suggest an interrelationship between hormone-binding capacity and the fluctuation of intracellular Ca2+ levels . Either hormone binding depends on the availability of Ca2+, or, alternatively, the latter depends on the binding capacity . Further studies are required to elucidate the true nature of this interdependence.

Int J Biochem, 1987, 19(8), 741 - 3
The specific activities of mesophilic and thermophilic proteinases; Cowan DA et al.; 1 . Most enzymes from extreme thermophiles do not possess higher specific activities than similar enzymes from mesophiles (measured at their respective growth temperatures) . 2 . However, using protein substrates, the specific activities of thermophilic proteinases are considerably higher than those of most microbial and eukaryotic proteinases . 3 . This property could be attributed to purely kinetic influences on the enzyme, to some specific "design" feature of the proteinase, or to the effects of temperature on the substrate . 4 . Comparisons of the rates of hydrolysis of large and small substrates by both mesophilic and thermophilic proteinases suggest that temperature-induced changes in substrate susceptibility are a major factor.

J Dairy Sci, 1987 Jan, 70(1), 1 - 12
Survival of lactic acid bacteria in the human stomach and adhesion to intestinal cells; Conway PL et al.; The survival of four strains of lactic acid bacteria in human gastric juice, in vivo and in vitro, and in buffered saline, pH 1 to 5, has been investigated . The strains studied include two Lactobacillus acidophilus strains, Lactobacillus bulgaricus, and Streptococcus thermophilus . In addition, the adhesion of these strains to freshly collected human and pig small intestinal cells and to pig large intestinal cells has been studied and the effect of milk on both survival and adhesion tested . As a result of these investigations, an in vitro test system for screening potential cultures for use as human dietary adjuncts can be developed . The ability to survive in gastric juice and to adhere varied significantly for the strains tested; L . acidophilus ADH survived and adhered better than the others while S . thermophilus survived and adhered poorly . For all strains, both survival and adhesion was enhanced by milk . As all strains adhered to some extent to both human and pig intestinal cells, the adhesion mechanism is probably a nonspecific attachment as opposed to other reported specific Lactobacillus adhesion to gastric tissue . From the survival and adhesion data it seems feasible to obtain elevated levels of viable Lactobacillus sp . in human intestine by careful selection of the bacterial strains ingested . Furthermore, the in vitro methods used here should be valuable to screen potential strains . The data presented here can then be correlated with human in vivo studies monitoring the beneficial effect of ingestion of these Lactobacillus.

Biochem J, 1987 Jan 1, 241(1), 285 - 90
The circular-dichroic properties of the 'Rieske' iron-sulphur protein in the mitochondrial ubiquinol: cytochrome c reductase; Degli Esposti M et al.; We have studied the c.d . spectra of the 'Rieske' iron-sulphur protein isolated from the ubiquinol: cytochrome c reductase (bc1 complex) of bovine heart mitochondria . Both the oxidized and the reduced form of the 'Rieske' protein display a series of well-resolved c.d . features resembling those reported for the 'Rieske'-type iron-sulphur protein purified from the bacterium Thermus thermophilus {Fee, Findling, Yoshida, Hille, Tarr, Hearshen, Dunham, Day, Kent & Munck (1984) J . Biol, Chem . 259, 124-133} . In particular, the difference spectra, reduced minus oxidized, of both proteins have a distinctive negative band at 497 nm . The c.d . features characteristic of the isolated 'Rieske' protein were found in the dichroic spectra of the whole bc1 complex in the region between 450 and 520 nm . The reduction of the enzyme by ascorbate or ubiquinol is accompanied by the formation of a negative band at about 500 nm that corresponds, in all its c.d . properties, to the specific dichroic absorption of the reduced 'Rieske' iron-sulphur protein.

Mol Cell Biol, 1987 Jan, 7(1), 435 - 43
Nucleotide sequence structure and consistency of a developmentally regulated DNA deletion in Tetrahymena thermophila; Austerberry CF et al.; DNA deletion by site-specific chromosome breakage and rejoining occurs extensively during macronuclear development in the ciliate Tetrahymena thermophila . We have sequenced both the micronuclear (germ line) and rearranged macronuclear (somatic) forms of one region from which 1.1 kilobases of micronuclear DNA are reproducibly deleted during macronuclear development . The deletion junctions lie within a pair of 6-base-pair direct repeats . The termini of the deleted sequence are not inverted repeats . The precision of deletion at the nucleotide level was also characterized by hybridization with a synthetic oligonucleotide matching the determined macronuclear (rejoined) junction sequence . This deletion occurs in a remarkably sequence-specific manner . However, a very minor degree of variability in the macronuclear junction sequences was detected and was shown to be inherent in the mechanism of deletion itself . These results suggest that DNA deletion during macronuclear development in T . thermophila may constitute a novel type of DNA recombination and that it can create sequence heterogeneity on the order of a few base pairs at rejoining junctions.

Gene, 1987, 54(1), 65 - 71
Nucleotide sequence of the phosphofructokinase gene from Bacillus stearothermophilus and comparison with the homologous Escherichia coli gene; French BA et al.; The gene (Bs-pfk) for phosphofructokinase (PFK) from Bacillus stearothermophilus has been cloned and sequenced . The deduced amino acid sequence is nearly identical to the sequence which was previously determined by peptide analysis . The elevated G + C content of Bs-pfk relative to the homologous Ec-pfkA from Escherichia coli is consistent with previous observations concerning genes from thermophilic prokaryotes . A significant degree of homology exists when the deduced amino acid sequence of B . stearothermophilus PFK is compared with the corrected sequences of rabbit muscle PFK or E . coli PFK-1 . The cloning and sequencing of Bs-pfk completes the first step toward using site-specific mutagenesis to investigate the structure-function relationships for this allosteric enzyme.

Gene, 1987, 55(2-3), 169 - 78
A restriction fragment length polymorphism in the 5' non-transcribed spacer of the rDNA of Tetrahymena thermophila inbred strains B and C3; Luehrsen KR et al.; The ribosomal DNA of Tetrahymena thermophila is a 21-kb palindromic molecule which replicates autonomously in the macronuclei of this species . In addition to the rRNA coding regions, there are 5' and 3' flanking sequences which are not transcribed (non-transcribed spacer; NTS) . The 5' NTS contains a bidirectional origin of DNA replication and promoter elements which direct transcription . We have identified a restriction fragment length polymorphism in the rDNA 5' NTS by comparing the B and C3 inbred strains of T . thermophila . There is a 42-bp region present in the C3 but not in the B strain rDNA; we present evidence that this difference most likely represents a deletion in the B strain rather than an insertion in the C3 strain . We also include a revised version of the nucleotide sequence of the 5' NTS DNA of inbred strain B.

Curr Genet, 1987, 11(5), 353 - 7
Yeast centromere sequences do not confer mitotic stability on circular plasmids containing ARS elements of Tetrahymena thermophila rDNA; Amin AA et al.; We have previously demonstrated that a 657 bp TaqI-XbaI and a 427 bp XbaI-XbaI fragment from the 5' non-transcribed spacer of the extrachromosomal ribosomal DNA of Tetrahymena thermophila function as autonomously replicating sequences (ARS) in Saccharomyces cerevisiae . These fragments are adjacent to each other in a region that encompasses the in vivo origin of bidirectional replication of rDNA . The presence of a yeast centromere (CEN) fragment does not confer mitotic stability on these plasmids . A sensitive yeast colony colour assay (Hieter et al . 1985a) has been used to evaluate the cis-acting effect of each ARS segment on the pattern of inheritance of a plasmid containing CEN5:URA3:SUP4 . Colonies of transformed cells obtained both in the presence and absence of selection were red with no detectable white or pink sectors . The lack of sectoring indicates that both plasmids are lost at an extremely high rate, likely due to 1:0 segregation events . We conclude that while these ARS elements confer a high frequency transformation phenotype, they lack a function which is required in cis for the maintenance of mitotic stability in the presence of a centromere . This missing cis-acting function may result in the inability of the plasmids to be brought under the control of cell-cycle regulated replication.

Gene, 1987, 56(2-3), 313 - 9
The telomeres of Tetrahymena ribosomal DNA are not sufficient for stabilizing linear DNA in Xenopus oocytes; Yu XM et al.; Restriction fragments that include the telomeres of ribosomal DNA from Tetrahymena thermophila (TtrDNA) were ligated to the ends of linearized simian virus 40 (SV40) DNA . The linear SV40 DNA with TtrDNA ends, circular SV40 DNA, linear SV40 DNA, and intact TtrDNA were injected into the nuclei of Xenopus laevis oocytes and assayed for stability . The intact linear 21-kb TtrDNA and circular SV40 DNA were maintained stably for at least 72 h after injection while the linearized SV40 DNA, either with or without telomeric ends, was degraded rapidly . Limited digestion with micrococcal nuclease revealed that neither the intact TtrDNA nor the SV40 DNA with telomeric ends reconstituted into chromatin containing regularly spaced nucleosomes . Another linearized plasmid DNA (pBamC), 14 kb in length, also was not stable in Xenopus oocytes with or without the addition of TtrDNA telomeres . Therefore, TtrDNA telomeres by themselves are not sufficient for stabilization of linear DNA in Xenopus oocytes . Rather, linear TtrDNA is maintained stably because of additional sequence or structural information encoded within the molecule.

Cytobios, 1987, 52(208), 17 - 22
Taxon-dependence of receptor level cell-to-cell communication in Tetrahymena: possible explanation for the transmission of hormonal imprinting; Csaba G et al.; Insulin treatment induced in Tetrahymena pyriformis a positive hormonal imprinting, and in Tetrahymena thermophila a negative imprinting, resulting in increased and decreased binding capacity, respectively, at re-exposure to the hormone . The imprinting, or the information associated with it, is transferred by the nutrient medium of the insulin-treated cells to those not treated . The issue of transfer depends on the nature of the receiver taxon, leading always to a positive imprinting in Tetrahymena pyriformis, and to a negative imprinting in Tetrahymena thermophila, regardless of the nature of the 'imprinted' transmitter taxon . The findings substantiate the transferability of hormonal imprinting by the nutrient medium at the unicellular level, the key role of the postreceptorial mechanism in determining the trend of imprinting and may explain the persistence of imprinting in the progeny generations.

Adv Biophys, 1987, 23, 115 - 47
Dynamic structures and functions of transfer ribonucleic acids from extreme thermophiles; Yokoyama S et al.; tRNA species from an extreme thermophile T . thermophilus that grows up to 85 degrees C have been found to be more thermostable than those from moderate thermophiles and mesophiles . Such thermostability of T . thermophilus tRNA species is partly due to the high contents of G.C base pairs in the stem regions . In addition, a novel modified nucleoside s2T has been found that substitutes T in position 54 . The extent of 2-thiolation of T(54) has been found to depend on environmental temperatures from 50 to 80 degrees C . Two tRNA(Ile) species have been isolated from T . thermophilus HB8, tRNA(1aIle) with s2T(54) and tRNA(1bIle) with T(54), which have the identical nucleotide sequence except for position 54 . However, the melting temperature of tRNA(1aIle) is higher by 3 degrees C than that of tRNA(1bIle) . This clearly indicates that the 2-thiolation of T(54) contributes directly to the thermostability of T . thermophilus tRNA species . Proton NMR analyses have shown that the nucleoside s2T is "rigid" and predominantly takes the C3'-endo-gg-anti form of A-RNA, because of the steric effect of the bulky 2-thiocarbonyl groups and the 2'-hydroxyl group . Thus, the inherent rigidity of s2T in position 54 significantly enhances the stability of the tertiary structure of tRNA . In protein synthesis of T . thermophilus, s2T(54)-bearing tRNA and T(54)-bearing tRNA species are selectively utilized depending on environmental temperature . In the anticodons of major tRNA species from T . thermophilus, G or C exclusively appears in the first position, and GGN and CCN are favored over synonymous GCN or CGN . These characteristic anticodon sequences correspond to the characteristic codon usage in thermophile genes.

Mol Biol Rep, 1987, 12(1), 13 - 9
Small RNAs of Tetrahymena thermophila; Horvath P et al.; Highly purified nuclear and cytoplasmic RNAs were obtained from Tetrahymena thermophila BVII containing only a minimal amount of cross-contamination . In the nuclear RNA fraction we have detected at least 6 distinct snRNAs . Some of the RNA species showed microheterogeneity . SnRNAs of Tetrahymena thermophila are very similar to rat snRNAs, as far as length is concerned . Our cytoplasmic small RNA fraction contained two RNAs, 7S and T7, reported recently as nuclear, particularly nucleolar RNAs . Moreover, we could detect only one cytoplasmic small RNA species Tc1, Tc2 was not observed . Neither the nuclear nor the cytoplasmic small RNA species are degradation products of ribosomal RNA as was shown by Northern blotting and following hybridization with pGY17 containing the entire transcribed region of the ribosomal DNA of Tetrahymena thermophila.

Nature, 1986 Dec 18-31, 324(6098), 695 - 7
A new way of enhancing the thermostability of proteases; Imanaka T et al.; Thermostability of enzyme can be enhanced by single amino acid substitutions . Recent advances in genetic engineering have made it possible to create novel proteins in a predictable manner where structural information for the protein is available . This 'protein engineering' has already been used to enhance enzyme thermostability, but it is usually not clear which amino acid substitutions should be made . We consider that the following approach should be helpful in engineering proteins with enhanced thermostability: highly conserved residues should be left unchanged; the sequences of known mesophilic and thermophilic proteins should be used to suggest the kinds of changes likely to increase thermostability; and substitutions should be made that increase internal hydrophobicity and that stabilize helices for strong internal packing . We describe here the use of this approach to alter the thermostability of the thermostable neutral protease of Bacillus stearothermophilus, the sequence of which is known . Surprisingly we find that a single mutation that decreases thermostability can require two mutations that increase stability to compensate for it . The effects on stability are not additive, suggesting cooperativity.

Mol Cell Biol, 1986 Dec, 6(12), 4742 - 4
Methylation site within a facultatively persistent sequence in the macronucleus of Tetrahymena thermophila; White TC et al.; DNA methylation occurs at the adenines in the somatic macronucleus of Tetrahymena thermophila . We report on a methylation site within a DNA segment showing facultative persistence in the macronucleus . When the site is present, methylation occurs on both strands, although only 50% of the DNA molecules are methylated.

Appl Environ Microbiol, 1986 Dec, 52(6), 1242 - 6
Heat resistance of bacterial spores correlated with protoplast dehydration, mineralization, and thermal adaptation; Beaman TC et al.; Twenty-eight types of lysozyme-sensitive spores among seven Bacillus species representative of thermophiles, mesophiles, and psychrophiles were obtained spanning a 3,000-fold range in moist-heat resistance . The resistance within species was altered by demineralization of the native spores to protonated spores and remineralization of the protonated spores to calcified spores and by thermal adaptation at maximum, optimum, and minimum sporulation temperatures . Protoplast wet densities, and thereby protoplast water contents, were obtained by buoyant density sedimentation in Nycodenz gradients (Nyegaard and Co., Oslo, Norway) . Increases in mineralization and thermal adaptation caused reductions in protoplast water content between limits of ca . 57 and 28% (wet weight basis), and thereby correlated with increases in sporal heat resistance . Above and below these limits, however, increases in mineralization and thermal adaptation correlated with increases in sporal resistance independently of unchanged protoplast water contents . All three factors evidently contributed to and were necessary for heat resistance of the spores, but dehydration predominated.

Eur J Biochem, 1986 Dec 1, 161(2), 263 - 71
Purification and characterization of propylamine transferase from Sulfolobus solfataricus, an extreme thermophilic archaebacterium; Cacciapuoti G et al.; The enzyme propylamine transferase, catalyzing the transfer of the propylamine moiety from S-adenosyl(5')-3-methylthiopropylamine to several amine acceptors, has been purified 643-fold in 20% yield from Sulfolobus solfataricus, an extreme thermophilic archaebacterium optimally growing at 87 degrees C . The purified enzyme (specific activity 2.05 units/mg protein), is homogeneous by criteria of gel electrophoresis, gel filtration, isoelectric focusing and ultracentrifugation analysis . The molecular mass of the native enzyme was estimated to be about 110 kDa by gel permeation and ultracentrifugation analysis . The protein migrates on SDS/polyacrylamide gel electrophoresis as a single band of 35 kDa, suggesting that the enzyme is a trimer composed by identical subunits . An optimum pH of 7.5 and an acidic isoelectric point of 5.3 have been calculated . The optimum temperature was 90 degrees C and no loss of activity is observable even after exposure of the purified enzyme to 100 degrees C for 1 h . No reducing agents are required for enzymatic activity . Substrate specificity towards the amine acceptors is rather broad in that 1,3-diaminopropane (Km = 1675 microM), putrescine (Km = 3850 microM), sym-norspermidine (Km = 954 microM) and spermidine (Km = 1539 microM) are recognized as substrates . Conversely S-adenosyl(5')-3-methylthiopropylamine is the only propylamine donor (Km = 7.9 microM) and the deamination of the sulfonium compound prevents the recognition by the enzyme . The reaction is irreversible and initial-rate kinetic studies indicate that the propylamine transfer is operated through a sequential mechanism . 5'-Methylthioadenosine, a product of the reaction, acts as a powerful competitive inhibitor with a Ki of 3.7 microM . Enzyme-substrate binding sites have been investigated with the aid of several substrate analogs and products . Among the compounds assayed, 5'-methylthiotubercidin, S-adenosyl(5')-3-thiopropylamine and S-adenosyl-3-thio-1,8-diaminooctane are the most active inhibitors.

J Bacteriol, 1986 Dec, 168(3), 1053 - 8
Isolation of an enzyme complex with carbon monoxide dehydrogenase activity containing corrinoid and nickel from acetate-grown Methanosarcina thermophila; Terlesky KC et al.; Fast protein liquid chromatography of cell extract from methanol- or acetate-grown Methanosarcina thermophila resolved two peaks of CO dehydrogenase activity . The activity of one of the CO dehydrogenases was sixfold greater in acetate-grown compared with methanol-grown cells . This CO dehydrogenase was purified to apparent homogeneity (70 mumol of methyl viologen reduced per min per mg of protein) and made up greater than 10% of the cellular protein of acetate-grown cells . The native enzyme (Mr 250,000) formed aggregates with an Mr of approximately 1,000,000 . The enzyme contained five subunits (Mrs 89,000, 71,000, 60,000, 58,000, and 19,000), suggesting a multifunctional enzyme complex . Nickel, iron, cobalt, zinc, inorganic sulfide, and a corrinoid were present in the complex . The UV-visible spectrum suggested the presence of iron-sulfur centers . The electron paramagnetic resonance spectrum contained g values of 2.073, 2.049, and 2.028; these features were broadened in enzyme that was purified from cells grown in the presence of medium enriched with 61Ni, indicating the involvement of this metal in the spectrum . The pattern of potassium cyanide inhibition indicated that cyanide binds at or near the CO binding site . The properties of the enzyme imply an involvement in the dissimilation of acetate to methane, possibly by cleavage of acetate or activated acetate.

J Biochem (Tokyo), 1986 Dec, 100(6), 1569 - 73
Ion permeability of ciliary membrane vesicles isolated from Tetrahymena . Single-channel recording study on the membrane reconstituted into a planar lipid bilayer; Kawahara S et al.; An ion channel in ciliary membrane vesicles isolated from Tetrahymena pyriformis NT-1 was characterized with a single-channel recording technique applied to the membrane reconstituted into a planar lipid bilayer made of soybean phospholipid . The gating of this channel is independent of membrane potential and it spends most of the time in its open-channel state . It is selective for metal cations over gluconate anion although it little distinguishes cations, Ca2+, Ba2+, Mg2+, and K+ . This channel seems to be identical to the monovalent cation channel in Tetrahymena thermophila BII cilia, which was recently reported by Oosawa and Sokabe (Am . J . Physiol . 249, C177-C179, 1985) . The channel conductance saturates with increasing ion concentration . The maximum value (in pS) for the single-channel conductance obtained in symmetric solutions of sufficiently high concentration is in the order of K+(212) greater than Ba2+(41) greater than Ca2+(22) greater than Mg2+(20) . On the other hand, the affinity of the channel for these ions is in the reverse order.

Biochemistry, 1986 Nov 4, 25(22), 7031 - 6
Conformation change of tRNAGlu in the complex with glutamyl-tRNA synthetase is required for the specific binding of L-glutamate; Hara-Yokoyama M et al.; The binding of Thermus thermophilus glutamyl-tRNA synthetase (GluRS) with T . thermophilus tRNAGlu, Escherichia coli tRNAGlu, and amino acids was studied by fluorescence measurements . In the absence of tRNAGlu, GluRS binds with D-glutamate as well as L-glutamate . However, in the presence of E . coli tRNAGlu, GluRS binds specifically with L-glutamate . The KCl effects on the Michaelis constants (Km) for tRNAGlu, L-glutamate, and ATP were studied for the aminoacylation of the homologous tRNAGlu and heterologous tRNAGlu species . As the KCl concentration is raised from 0 to 100 mM, the Km value for L-glutamate in the heterologous system is remarkably increased whereas the Km value for L-glutamate in the homologous system is only slightly increased . The circular dichroism analyses were made mainly of the bands due to the 2-thiouridine derivatives of tRNAGlu in the complex . The conformation change of T . thermophilus tRNAGlu upon complex formation with GluRS is not affected by addition of KCl . In contrast, the heterologous tRNAGlu X GluRS complex is in an equilibrium of two forms that depends on KCl concentration . The predominant form at low KCl concentration is closely related to the small Km value for L-glutamate . In this form of the complex, the conformation of tRNAGlu is appreciably different from that of free molecule . Accordingly, such a conformation change of tRNAGlu in the complex with GluRS is required for the specific binding of L-glutamate as the substrate.

Mol Cell Biol, 1986 Nov, 6(11), 3854 - 61
Characterization of a Tetrahymena thermophila mutant strain unable to develop normal thermotolerance; Kraus KW et al.; For Tetrahymena thermophila cells to survive extended periods of time at 43 degrees C, they must continuously synthesize heat shock proteins . For its translational machinery to function at 43 degrees C, T . thermophila requires either prior nonlethal heat shock treatment or brief treatment with partially inhibiting doses of cycloheximide or emetine . We have identified and characterized a mutant strain of T . thermophila (MC-3) in which prior nonlethal heat shock does not prevent protein synthesis inactivation at 43 degrees C . In addition, treatment of MC-3 cells with either of the antibiotics that normally confer 43 degrees C thermoprotection on wild-type cells elicited no similar thermoprotective response in these cells . Despite these phenotypic characteristics, by other criteria MC-3 synthesized a normal, functional array of heat shock proteins at 40 degrees C, a nonlethal heat shock protein-inducing temperature . The mutation in MC-3 which prevents the thermostabilization of protein synthesis by nonlethal heat shock is, by genetic criteria, most likely the same one which prevents the induction of thermotolerance by drug treatments . We present evidence that this mutation may affect some ribosome-associated functions.

J Bacteriol, 1986 Nov, 168(2), 563 - 7
Characterization of a manganese-containing catalase from the obligate thermophile Thermoleophilum album; Allgood GS et al.; A manganese-containing catalase has been characterized from Thermoleophilum album NM, a gram-negative aerobic bacterium obligate for thermophily and n-alkane substrates . The level of catalase in cells was increased about ninefold by growth in the presence of paraquat (2.5 microM), a superoxide-generating toxicant . Superoxide dismutase levels were unaffected by this compound . The enzyme was purified from cultures grown in the presence of paraquat to greater than 95% homogeneity and had an Mr of 141,000 . The enzyme was composed of four subunits, and each had an Mr of 34,000 . There were 1.4 +/- 0.4 atoms of manganese present per subunit . The catalase had a Km for hydrogen peroxide of 15 mM and a Vmax of 11 mM/mg . Peroxidase activity, as measured with p-phenylenediamine, copurified with the catalase . Inhibitors of heme-catalase were weak inhibitors of the T . album enzyme . The optimum pH for catalase activity was 8 to 9 . The enzyme was stable from pH 6.5 to 11 and retained activity at assay temperatures from 25 to 80 degrees C . The catalase was stable for 24 h of incubation at 60 degrees C.

Proc Natl Acad Sci U S A, 1986 Nov, 83(22), 8674 - 8
An intervening sequence in an unusual histone H1 gene of Tetrahymena thermophila; Wu M et al.; An intervening sequence of 254 base pairs interrupts the coding region of the single gene for macronuclear histone H1 of the ciliated protozoan, Tetrahymena thermophila . The intervening sequence has splice junctions similar to those found in RNA polymerase II genes of other organisms . No obvious similarities are observed between this intron and the self-splicing intervening sequence of the Tetrahymena ribosomal gene . The derived amino acid sequence describes a small extremely basic H1 protein missing most of the central hydrophobic domain that is conserved in all other H1 proteins . Macronuclei divide amitotically, without chromosome condensation, suggesting the conserved globular domain of H1 plays a role in higher-order chromatin structure.

Proc Natl Acad Sci U S A, 1986 Nov, 83(21), 8221 - 5
Conjugation rescue of an exocytosis-competent membrane microdomain in Tetrahymena thermophila mutants; Satir BH et al.; Conjugation-rescue experiments with two Tetrahymena thermophila mutants (exo-) incapable of exocytosis (SB255, SB258) have been used to dissect regulatory steps in assembly of a functional membrane microdomain, the fusion rosette . "Rescue" refers to the recovery of a secretory activity . Exo- mutants fail to secrete mucus normally (form capsules) when stimulated by the secretagogue alcian blue and are blocked before the assembly of a functional fusion rosette in the cell membrane . Two criteria are used to assay recovery of the wild-type (exo+) phenotype: the conjugant's ability to form capsules when stimulated and the presence of assembled rosettes, which disperse upon stimulation . Conjugation of exo+ X SB258 results in restoration of secretion in 60% of the mutant conjugants and reappearance of assembled rosettes . Secretory capacity is restored in the SB258 cell within one-half hour of firm pair formation . This restoration is not due to new gene expression or continued protein synthesis, since it occurs when SB258 is crossed to a "star" strain (A*), which has defective micronuclei and therefore cannot contribute wild-type genes, and restoration occurs in the presence of cycloheximide during conjugation . Conjugation of exo+ X SB255 reveals a real but inefficient restoration of exocytic capacity in the exo- conjugant and a significant decrease of exocytic capacity in the exo+ conjugant . SB255 X SB258 crosses also show a low but significant rescue of exocytic competence, indicating that different components of the exocytic mechanism are affected in the two mutants . This cross leads to restoration of rosette assembly and function in one of the partners, presumably SB258 . These results provide data about some of the steps necessary for rosette assembly and suggest that transferable factors that promote and/or inhibit exocytosis are present in these cells.

J Dairy Res, 1986 Nov, 53(4), 631 - 7
Influence of mastitis on growth of starter organisms used for the manufacture of fermented milks; Okello-Uma I et al.; The effect of milk containing increased somatic cells on the starter organisms Streptococcus thermophilus, Lactobacillus bulgaricus and L . acidophilus was examined . Increased somatic cell count resulted in stimulation of Str . thermophilus owing to increased proteolysis, but inhibition of L . acidophilus as a consequence of increased phagocytic activity of the polymorphonuclear leucocytes

Biochim Biophys Acta, 1986 Oct 29, 884(1), 182 - 90
Spin-labelled analogues of GDP and GTP as site-specific reporter groups for guanosine nucleotide-binding proteins; Faulhammer HG et al.; New derivatives of GDP and GTP have been synthesized for the spectroscopic investigation of the interaction between guanosine nucleotides and guanosine nucleotide-binding proteins . The 3'-hydroxyl group in these nucleotides was replaced by a 3'-amino group, which was further derivatized by the introduction of a spin-label reporter group . The biological activity of 3'SL-GDP and 3'SL-GTP could be demonstrated by measuring the interaction of these spin-labelled derivatives with bacterial elongation factor Tu . The amino modification and spin labelling only slightly influenced the affinity of the guanosine nucleotides for EF-Tu from Escherichia coli or Thermus thermophilus . Electron paramagnetic resonance (EPR) measurements revealed a strong immobilization of the labelled nucleotides upon binding to T . thermophilus EF-Tu . Significant differences between the spectra of EF-Tu X 3'SL-GDP, EF-Tu X 3'SL-GTP and aminoacyl-tRNA X EF-Tu X 3'SL-GTP ternary complexes were observed . Our data demonstrate that spin-labelled guanosine nucleotides can be used as sensitive spectroscopic probes for the investigation of the local environment of the nucleotide-binding site during distinct functional states of a guanosine nucleotide-binding protein.

J Biol Chem, 1986 Oct 25, 261(30), 14178 - 83
Nucleotide sequence of the malate dehydrogenase gene of Thermus flavus and its mutation directing an increase in enzyme activity; Nishiyama M et al.; The nucleotide sequence of the malate dehydrogenase (mdh) gene from a thermophilic bacterium, Thermus flavus, was determined . The amino acid sequence of the Thermus malate dehydrogenase resembled that of the porcine heart cytoplasmic enzyme to a certain extent, and Asp-159 and His-187 were identified as possible essential residues for the catalytic function . The mutated mdh gene was also cloned from a spontaneous mutant of T . flavus containing a higher activity of the enzyme . Its mutation point was determined to be a single nucleotide exchange from C to T which caused Thr-190 to be substituted by isoleucine . The mutated enzyme showed resistance to substrate inhibition, an increase in both kcat and Km, and a shift toward a more acid optimum pH for the enzyme reaction.

Eur J Biochem, 1986 Oct 15, 160(2), 433 - 40
Nucleotide sequence and characteristics of the gene for L-lactate dehydrogenase of Thermus caldophilus GK24 and the deduced amino-acid sequence of the enzyme; Kunai K et al.; The gene for L-lactate dehydrogenase (LDH) (EC 1.1.1.27) of Thermus caldophilus GK24 was cloned in Escherichia coli using synthetic oligonucleotides as hybridization probes . The nucleotide sequence of the cloned DNA was determined . The primary structure of the LDH was deduced from the nucleotide sequence . The deduced amino acid sequence agreed with the NH2-terminal and COOH-terminal sequences previously reported and the determined amino acid sequences of the peptides obtained from trypsin-digested T . caldophilus LDH . The LDH comprised 310 amino acid residues and its molecular mass was determined to be 32,808 . On alignment of the whole amino acid sequences, the T . caldophilus LDH showed about 40% identity with the Bacillus stearothermophilus, Lactobacillus casei and dogfish muscle LDHs . The T . caldophilus LDH gene was expressed with the E . coli lac promoter in E . coli, which resulted in the production of the thermophilic LDH . The gene for the T . caldophilus LDH showed more than 40% identity with those for the human and mouse muscle LDHs on alignment of the whole nucleotide sequences . The G + C content of the coding region for the T . caldophilus LDH was 74.1%, which was higher than that of the chromosomal DNA (67.2%) . The G + C contents in the first, second and third positions of the codons used were 77.7%, 48.1% and 95.5% respectively . The high G + C content in the third base caused extremely non-random codon usage in the LDH gene . About half (48.7%) the codons in the LDH gene started with G, and hence there were relatively high contents of Val, Ala, Glu and Gly in the LDH . The contents of Pro, Arg, Ala and Gly, which have high G + C contents in their codons, were also high . Rare codons with U or A as the third base were sometimes used to avoid the TCGA sequence, the recognition site for the restriction endonuclease, TaqI . Two TCGA sequences were found only in the sequence of CTCGAG (XhoI site) in the sequenced region of the T . caldophilus DNA . There were three segments with similar sequences in the two 5' non-coding regions, probably the promoter and ribosome-binding regions, of the genes for the T . caldophilus LDH and the Thermus thermophilus 3-isopropylmalate dehydrogenase.

Cell, 1986 Oct 10, 47(1), 73 - 80
Evidence in favor of the symbiotic origin of chloroplasts: primary structure and evolution of tobacco glyceraldehyde-3-phosphate dehydrogenases; Shih MC et al.; We report nucleotide sequences of cDNAs for the nuclear genes encoding chloroplast (GapA and GapB) and cytosolic (GapC) glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from N . tabacum . Comparison of nucleotide sequences indicates that the GapA and GapB genes evolved following duplication of an ancestral gene about 450 million years ago . However, the divergence of GapA/B and GapC occurred much earlier in evolution than the divergence of GapC and GAPDH genes of animals and fungi, suggesting that chloroplast and cytosolic GAPDHs evolved from different lineages . Comparison of amino acid sequences shows that the chloroplast GAPDHs are related to GAPDHs found in thermophilic bacteria, while the cytosolic GAPDH is related to the GAPDH found in mesophilic prokaryotes . These results strongly support the symbiotic origin of chloroplasts.

Nucleic Acids Res, 1986 Oct 10, 14(19), 7597 - 615
In situ dot blots: quantitation of mRNA in intact cells; Yu SM et al.; A rapid, simple and reproducible dot blot method is described for quantitating the amounts of specific messages in small numbers of intact cells . The method has been used to accurately determine the number of histone H4 mRNA molecules in growing (approximately 40,000) and in starved (approximately 1600) Tetrahymena thermophila, and to measure the amount of message contributed by an E . coli plasmid containing part of the S10 ribosomal operon . Use of the method is illustrated to optimize in situ hybridization protocols and to measure mRNA amounts in cell lysates . Preliminary studies also indicate that the method can be used to detect mRNA in intact yeast cells.

Biochim Biophys Acta, 1986 Oct 3, 878(3), 450 - 3
Effect of dimethylaminoethylphosphonate on phospholipid metabolism in Tetrahymena; Smith JD; Dimethylaminoethylphosphonate (DMAEP) was incorporated into the phospholipids of the ciliate protozoan Tetrahymena thermophila at the expense of both phosphatidylethanolamine and phosphatidylcholine, but it had no effect on the levels of the 2-aminoethylphosphonolipid . The newly formed DMAEP-lipid accounted for almost 50% of the phospholipids of the organism . The DMAEP was incorporated into the phospholipids using both the ethanolaminephosphotransferase and cholinephosphotransferase pathways . The DMAEP-lipid was not methylated to the trimethyl derivative, confirming the lack of methylation of phosphonolipids by Tetrahymena.

J Dairy Sci, 1986 Oct, 69(10), 2569 - 76
Improved media for differentiation of rods and cocci in yogurt; Matalon ME et al.; Selected yogurt starters and commercial samples grew on Elliker's lactic agar supplemented with .1% Tween 80 and 50 micrograms/ml of 2,3,5-triphenyltetrazolium chloride to produce small, red Streptococcus thermophilus colonies and larger, white Lactobacillus bulgaricus colonies . The distinction was somewhat strain dependent but was satisfactory in most cases . Addition of 7% skim milk (11% solids) to lactic agar in place of 2,3,5-triphenyltetrazolium chloride allowed good rod-coccus differentiation, regardless of strain or yogurt brand . On this medium, called yogurt lactic agar, L . bulgaricus appeared as large white colonies surrounded by a cloudy zone and S . thermophilus as smaller white colonies devoid of a surrounding halo . Casein precipitation was responsible for the halo effect around the more acidogenic L . bulgaricus colonies . Yogurt lactic agar compared favorably with S . thermophilus and Lactobacillus agar media for the recovery of S . thermophilus and L . bulgaricus in single and mixed cultures.

J Dairy Sci, 1986 Oct, 69(10), 2558 - 68
Influence of temperature on associative growth of Streptococcus thermophilus and Lactobacillus bulgaricus; Radke-Mitchell LC et al.; Compatibility of Streptococcus thermophilus and Lactobacillus bulgaricus during associative growth as dependent on optimum growth temperature was determined . Optimum growth temperatures for 9 strains of S . thermophilus and 10 strains of L . bulgaricus ranged from 35 to 42 degrees C for S . thermophilus and 43 to 46 degrees C for L . bulgaricus . Streptococcus thermophilus and L . bulgaricus strains exhibiting similar to divergent optimum growth temperatures were combined (1:1 vol/vol) and incubated in milk at 37, 42, and 45 degrees C until pH 4.2 was reached . Initial and postincubation cell numbers were determined by plate count method . Streptococcus thermophilus strains reached greater cell numbers than L . bulgaricus at 37, 42, and 45 degrees C in 93.3% of the mixed culture trials . Average rod-coccus ratios obtained at 37, 42, and 45 degrees C were 1:2.2, 1:8, and 1:2.4, respectively . Optimum growth temperatures had no influence on growth of L . bulgaricus and S . thermophilus in mixed culture . Rather, temperature appeared to influence compatibility by determining the concentration or type of stimulatory factor(s) produced by L . bulgaricus . All strains of S . thermophilus exhibited an uncoupling of growth from acid production . Optimum temperature for acid production ranged from 2 to 8 degrees C above optimum growth temperature . These findings warrant consideration in the manufacture of yogurt and other fermented milk products.

J Dairy Sci, 1986 Oct, 69(10), 2583 - 8
A comparison of beta-galactosidase specific activities in strains of Streptococcus thermophilus; Occhino LA et al.; Six Streptococcus thermophilus strains were examined for growth, acid production, and beta-galactosidase activity per milligram protein (specific activity), so that strain comparisons could be made . A wide range in activity was observed . Activity depended on growth time in M17 broth and, for most strains, continued to increase after cells had reached stationary phase . Maximum activity was at 16 h and ranged from 0 to 58 units/mg protein . Strain ST exhibited no beta-galactosidase activity but had trace phospho-beta-galactosidase activity (.8 units/mg protein after 2 h of growth) . Strains 3641 and TS2B exhibited slower growth rates and lower beta-galactosidase activities in milk as compared to M17 broth . Further, strain 3641 exhibited 10 times the activity of strain TS2B (2.86 vs . .24 units) after 4 h of growth in milk.

Eur J Biochem, 1986 Oct 1, 160(1), 37 - 40
1H and 13C NMR assignment of benzothiophenquinones from the sulfur-oxidizing archaebacterium Sulfolobus solfataricus; Lanzotti V et al.; From Sulfolobus solfataricus, a sulfur-oxidizing thermophilic member of archaebacteria, three unusual benzothiophenquinones were isolated . Detailed NMR studies on these quinones, including multipulse mono-dimensional and two-dimensional techniques, were performed to obtain carbon and proton assignments, one-bond, geminal and vicinal coupling constants and T1 relaxation times . This report extends the known quinone composition of thermophilic archaebacteria and further supports the concept that these biomolecules can serve as a useful chemotaxonomic tool.

J Bacteriol, 1986 Oct, 168(1), 318 - 21
Purification and characterization of an inorganic pyrophosphatase from the extreme thermophile Thermus aquaticus; Verhoeven JA et al.; An inorganic pyrophosphatase was purified over 600-fold to homogeneity as judged by polyacrylamide gel electrophoresis . The enzyme is a tetramer of Mr = 84,000, has a sedimentation coefficient of 5.8S, a Stokes radius of 3.5 nm, and an isoelectric point of 5.7 . Like the enzyme of Escherichia coli, the pyrophosphatase appears to be made constitutively . The pH and temperature optima are 8.3 and 80 degrees C, respectively . The Km for PPi is 0.6 mM . A divalent cation is essential, with Mg2+ preferred . The enzyme uses only PPi as a substrate.

J Biochem (Tokyo), 1986 Oct, 100(4), 923 - 34
Stable structure of thermophilic proton ATPase beta subunit; Kagawa Y et al.; F1-ATPase is the major enzyme for ATP synthesis in mitochondria, chloroplasts, and bacterial plasma membranes . F1-ATPase obtained from thermophilic bacterium PS3 (TF1) is the only ATPase which can be reconstituted from its primary structure . Its beta subunit constitutes the catalytic site, and is capable of forming hybrid F1's with E . coli alpha and gamma subunits . Since the stability of TF1 resides in its primary structure, we cloned a gene coding for TF1, and the primary structure of the beta subunit was deduced from the nucleotide sequence of the gene to compare the sequence with those of beta's of three major categories of F1's; prokaryotic membranes, chloroplasts, and mitochondria . The following results were obtained . Homology: The primary structure of the TF1 beta subunit (473 residues, Mr = 51,995.6) showed 89.3% homology with 270 residues which are identical in the beta subunits from human mitochondria, spinach chloroplasts, and E . coli . It contained regions homologous to several nucleotide-binding proteins . Secondary structure: The deduced alpha-helical (30.1%) and beta-sheet (22.3%) contents were consistent with those determined from the circular dichroism spectra . Residues forming reverse turns (Gly and Pro) were highly conserved among the F1 beta subunits . Substituted residues and stability of TF1: We compared the amino acid sequence of the TF1 beta subunit with those of the other F1 beta subunits mentioned above . The observed substitutions in the thermophilic subunit increased its propensities to form secondary structures, and its external polarity to form tertiary structure . Codon usage: The codon usage of the TF1 beta gene was found to be unique . The changes in codons that achieved these amino acid substitutions were much larger than those caused by minimal mutations, and the third letters of the optimal codons were either guanine or cytosine, except in codons for Gln, Lys, and Glu.

Nucleic Acids Res, 1986 Sep 25, 14(18), 7175 - 88
Ser-tRNAs from bovine mitochondrion form ternary complexes with bacterial elongation factor Tu and GTP; Gebhardt-Singh E et al.; Transfer ribonucleic acids were isolated from mitochondria of bovine heart and aminoacylated in vitro by a crude mitochondrial enzyme . Ser-tRNASerUCN and Ser-tRNASerAGY were isolated and characterized by partial sequencing . Although these tRNAs possess unique structural features not found in any bacterial tRNA, they form a ternary complex with elongation factor from the extreme thermophilic bacterium Thermus thermophilus and GTP.

Cell, 1986 Sep 12, 46(6), 873 - 83
The telomeres of the linear mitochondrial DNA of Tetrahymena thermophila consist of 53 bp tandem repeats; Morin GB et al.; We have cloned and sequenced the telomeric DNA of the linear mitochondrial DNA (mtDNA) of T . thermophila BVII . The mtDNA telomeres consist of a 53 bp sequence tandemly repeated from 4 to 30 times, with most molecules having 15 +/- 4 repetitions . The previously recognized terminal heterogeneity of the mtDNA is completely accounted for by the variability in the number of repeats . The 53 bp repeat does not resemble known telomeric DNA in sequence, repeat size, or number of repetitions . The termini occur at heterogeneous positions within the 53 bp repeat . The junction of the telomeric repeat with the internal DNA is at a different position within the telomeric repeat on each end of the mtDNA . We propose a model for the maintenance of the mtDNA ends involving unequal homologous recombination.

Exp Cell Res, 1986 Sep, 166(1), 161 - 70
The energy budget of Tetrahymena and the material fluxes into and out of the adenylate pool; Jauker F et al.; The material budget of the adenylate pool deals with all processes which physically establish and maintain this pool, while the energy budget is concerned with the intracompartmental ATP recycling . Both budgets were analysed in Tetrahymena thermophila exposed to various energy and material demands . Some of the general conclusions are: at a maximum growth rate the overall ATP consumption during one cell cycle is 10(-10) mol ATP; the contribution of osmoregulation and ciliary motion to the budget is about 1% each; at zero net growth, energy is consumed because of a continuous recycling of matter between the monomer and the polymer compartment . The rate of ATP production is about 1000-fold greater than the rate of adenylate monomer influx . The residence time of adenylate monomers within the pool is about 30 min, but for ATP molecules it is only 2 sec.

Mol Cell Biol, 1986 Sep, 6(9), 3240 - 5
Structure and expression of two temperature-specific surface proteins in the ciliated protozoan Tetrahymena thermophila; Bannon GA et al.; The presence of specific proteins (known as immobilization antigens) on the surface of the ciliated protozoan Tetrahymena thermophila is under environmental regulation . There are five different classes (serotypes) of surface proteins which appear on the cell surface when T . thermophila is cultured under different conditions of temperature or incubation medium; three of these are temperature dependent . The appearance of these proteins on the cell surface is mutually exclusive . We used polyclonal antibodies raised against 30 degrees C (designated SerH3)- and 40 degrees C (designated SerT)-specific surface antigens to study their structure and expression . We showed that these surface proteins contain at least one disulfide bridge . On sodium dodecyl sulfate-denaturing polyacrylamide gels, the nonreduced 30 degrees C- and 40 degrees C-specific surface proteins migrated with molecular sizes of 69 and 36 kilodaltons, respectively . The reduced forms of the proteins migrated with molecular sizes of 58 and 30 kilodaltons, respectively . The synthesis of the surface proteins responded rapidly and with a time course similar to that of the incubation temperature . The synthesis of each surface protein was greatly reduced within 1 h and undetectable by 2 h after a shift to the temperature at which the protein is not expressed . Surface protein synthesis resumed by the end of 1 h after a shift to the temperature at which the protein is expressed . The temperature-dependent induction of these surface proteins appears to be dependent on the synthesis of new mRNA, as indicated by a sensitivity to actinomycin D . Surface protein syntheses were mutually exclusive except at a transition temperature . At 35 degrees C both surface proteins were synthesized by a cell population . These data support the potential of this system as a model for the study of the effects of environmental factors on the genetic regulation of cell surface proteins.

J Biochem (Tokyo), 1986 Sep, 100(3), 797 - 808
Purification and immunofluorescence localization of the mutant gene product of a Tetrahymena cdaA1 mutant affecting cell division; Ohba H et al.; A division arrest mutant, cdaA, of Tetrahymena thermophila is known to have a ts-defect in the formation of the fission zone which determines the position of the fission plane . A protein (Mr = 85,000; pI = 4.7, designated as p85) has recently been identified in our laboratory as a possible gene product of the cdaA locus by two-dimensional gel electrophoresis and genetic experiments (Ohba et al., submitted) . In the present research, we have isolated p85, prepared its antibody, and demonstrated that in wild-type cells or in cdaA cells at permissive temperature, immunofluorescence for p85 appears on the equatorial basal bodies at the predicted fission zone just before formation of the zone . In such a case, the fission zone appears to be formed just anterior to the fluorescence-associated basal bodies, and then constriction of the division furrow occurs at the zone . However, in cdaA cells at the restrictive temperature, the equatorial p85 deposit and subsequent fission zone formation and furrowing do not occur at all . Thus, we conclude that p85 plays a key role in the formation of the fission zone and in the positioning of the equatorial fission line.

Appl Environ Microbiol, 1986 Sep, 52(3), 498 - 503
Method for determining virus inactivation during sludge treatment processes; Traub F et al.; A simple and reliable method is described which allows determination of virus inactivation rates during sludge treatment processes in situ . Bacteriophage f2 was adsorbed onto an electropositive membrane filter which was then sandwiched between two polycarbonate membranes with pores smaller than the virus diameter . The resulting sandwich was fixed in an open filter holder, and several such devices were connected before being exposed in sludge-digesting tanks . The device described prevented uncontrolled virus escape, but allowed direct contact of the various inactivating or stabilizing substances present in the environment tested with the virus adsorbed to the carrier membrane . After exposure to an environment, the surviving fraction of virus was eluted from the inner filter and determined by plaque counting . By using polycarbonate membranes without pores for sandwiching, the influence of temperature alone on virus inactivation could be measured . Thermophilic fermentation at 60 degrees C and at 65 kPa pressure led to a bacteriophage f2 titer reduction of 3.5 log10 units per h, whereas during thermophilic digestion at 54.5 degrees C titers decreased 1.2 log10 units per h . During mesophilic digestion an inactivation rate of only 0.04 log10 units per h was observed . Under these latter conditions, temperature had only a minor effect (19%) on virus inactivation, whereas at 54.5 degrees C during thermophilic digestion heat accounted for 32% of the total inactivation, and during thermophilic fermentation at 60 degrees C temperature and pressure were 100% responsible for virus denaturation.

Eur J Biochem, 1986 Sep 1, 159(2), 323 - 31
Prokaryotic features of a nucleus-encoded enzyme . cDNA sequences for chloroplast and cytosolic glyceraldehyde-3-phosphate dehydrogenases from mustard (Sinapis alba); Martin W et al.; Two cDNA clones, encoding cytosolic and chloroplast glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from mustard (Sinapis alba), have been identified and sequenced . Comparison of the deduced amino acid sequences with one another and with the GAPDH sequences from animals, yeast and bacteria demonstrates that nucleus-encoded subunit A of chloroplast GAPDH is distinct from its cytosolic counterpart and the other eukaryotic sequences and relatively similar to the GAPDHs of thermophilic bacteria . These results are compatible with the hypothesis that the nuclear gene for subunit A of chloroplast GAPDH is of prokaryotic origin . They are in puzzling contrast with a previous publication demonstrating that Escherichia coli GAPDH is relatively similar to the eukaryotic enzymes {Eur . J . Biochem . 150, 61-66 (1985)}.

Biophys J, 1986 Sep, 50(3), 457 - 61
Phospholipid surface bilayers at the air-water interface . III . Relation between surface bilayer formation and lipid bilayer assembly in cell membranes; Gershfeld NL; Lipid bilayer assembly in cell membranes has been simulated with total lipid extracts from human red blood cells and from mesophilic and thermophilic bacteria grown at several temperatures . Aqueous dispersions of these natural lipid mixtures form surface bilayers, a single bimolecular lipid state, but only at the growth temperature of the source organism . Thus, a single isolated bilayer state forms spontaneously in vitro from lipids that are available in vivo at the growth temperature of the cell . Surface bilayers form at a specific temperature that is a function of hydrocarbon chain length and degree of fatty acid unsaturation of the phospholipids; this property is proposed as an essential element in the control of membrane lipid composition.

Biol Chem Hoppe Seyler, 1986 Sep, 367(9), 891 - 903
Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria, IV . The primary structure of the mesophilic lactate dehydrogenase from Bacillus subtilis; Hediger MA et al.; The complete amino-acid sequence of lactate dehydrogenase from the mesophilic Bacillus subtilis (B . X1) was determined . Approximately 70% of the sequence was obtained by sequence analysis of intact protein (N-terminal sequence) and of four CNBr fragments (CNBr3, CNBr4, CNBr5 and CNBr6) . Sequences overlapping the CNBr fragments were determined from polypeptide fragments obtained by cleavage using o-iodosobenzoic acid (cleavage at Trp) or clostripain (cleavage at Arg) . The C-terminal amino-acid residue (Asn) was detected by carboxypeptidase Y-degradation . Lactate dehydrogenase from B . subtilis shows a 69% sequence homology to that from the thermophilic strain B . stearothermophilus, and a 34% sequence homology to those from higher organism . The homology of these enzymes is particularly high at the active site regions (the coenzyme and substrate binding sites) . The relatively high sequence conservation of the lactate dehydrogenases from B . subtilis and B . stearothermophilus (and from other bacilli) allows a structural comparison of this temperature variants.

Nature, 1986 Sep 25-Oct 1, 323(6086), 356 - 8
Cumulative effect of intragenic amino-acid replacements on the thermostability of a protein; Matsumura M et al.; The marginal net stability of a folded protein is thought to depend on a small difference between large, compensating individual forces . Therefore, the net free energy of stabilization of proteins is unexpectedly small (approximately 10 kcal mol-1) . The contribution of individual forces such as hydrogen bonds and salt bridges to the stabilization is evaluated as 1-3 kcal mol-1, and several additional forces are thought to be sufficient to account for the extra thermostability of thermophilic proteins . The native conformation of a protein is determined by the total number of interatomic interactions and hence by the amino-acid sequence . If the few amino-acid residues that individually contribute to the stabilization could be implemented concurrently into the sequence, the multiple replacement would enhance the overall stability of the protein molecule . Here we report evidence to support this argument . Thermal inactivation kinetics and proteolytic resistance for mutants of a kanamycin nucleotidyltransferase reveal that a few intragenic amino-acid replacements stabilize the protein cumulatively . Our experiments not only demonstrate the feasibility of elevating the thermostability of a protein but also lead to better understanding of the forces that are responsible for protein stability.

J Biochem (Tokyo), 1986 Sep, 100(3), 663 - 70
ADP binding to TF1 and its subunits induces ultraviolet spectral changes; Hisabori T et al.; Adenine nucleotide binding sites on the coupling factor ATPase of thermophilic bacterium PS3 (TF1) were investigated by UV spectroscopy and by equilibrium dialysis . When ADP was mixed with TF1 in the presence and in the absence of Mg2+, an UV absorbance change was induced (t1/2 approximately 1 min) with a peak at about 278 nm and a trough at about 250 nm . Similar spectral changes were induced by ADP with the isolated beta subunits in the presence and in the absence of Mg2+, and with the isolated alpha subunits in the presence of Mg2+ although the magnitudes of the changes were different . From equilibrium dialysis measurement we identified two classes of nucleotide binding sites in TF1 in the presence of Mg2+, three high-affinity sites (Kd = 61 nM) and three low-affinity sites (Kd = 87 microM) . In the absence of Mg2+, TF1 has one high-affinity site (Kd less than 10 nM) and five low-affinity sites (Kd = 100 microM) . Moreover, we found a single Mg2+-dependent ADP binding site on the isolated alpha subunit and a single Mg2+-independent ADP binding site on the isolated beta subunit . From the above observations, we concluded that the three Mg2+-dependent high-affinity sites for ADP are located on the alpha subunit in TF1 and that the single high-affinity site is located on one of the beta subunits in TF1 in the absence of Mg2+.

Arch Biochem Biophys, 1986 Aug 15, 249(1), 1 - 7
Functional linkage between phycobilisome and reaction center in two phycobilisome oxygen-evolving photosystem II preparations isolated from the thermophilic cyanobacterium Synechococcus sp; Kura-Hotta M et al.; Photosystem II oxygen-evolving preparations with attached phycobilisomes were isolated from the thermophilic cyanobacterium Synechococcus sp . with beta-octylglucoside or digitonin . Fluorescence emission spectra of the two preparations determined at 77 K largely lacked a far red band which originates from photosystem I . The spectrum of the digitonin preparation was otherwise similar to that of intact cells, whereas the beta-octylglucoside preparation showed a pronounced band at 687 nm, which is considered to be emitted from phycobilisomes . The relative yield of phycobilin fluorescence was similar between the digitonin preparations and the cells but was considerably larger in the beta-octylglucoside preparations at room temperature . The quantum yield of ferricyanide photoreduction determined with light which is absorbed mainly by phycobiliproteins was 0.85 for the digitonin preparation and 0.57 for the beta-octylglucoside preparation . The results indicate that excitation energy is transferred from phycobilisomes to photosystem II reaction centers in the digitonin preparation as efficiently as in intact cells, while a significant portion of light energy harvested by phycobilisomes is not utilized by the primary photochemistry in the beta-octylglucoside preparation . Digitonin and beta-octylglucoside preparations had 65 and 48 chlorophyll a molecules per photosystem II reaction center, respectively . The beta-octylglucoside preparation contained twice as much phycocyanin and allophycocyanin per photosystem II reaction center as the digitonin preparation, which has a phycobiliprotein-to-photosystem II reaction center ratio very similar to that of cells . It is concluded that whereas the beta-octylglucoside preparation contains a considerable amount of free phycobilisomes, all phycobilisomes present in the digitonin preparation are physically and functionally linked to photosystem II reaction center complexes.

Nucleic Acids Res, 1986 Aug 11, 14(15), 6299 - 311
Conservation of sequences adjacent to the telomeric C4A2 repeats of ciliate macronuclear ribosomal RNA gene molecules; Challoner PB et al.; We sequenced and compared the telomeric regions of linear rDNAs from vegetative macronuclei of several ciliates in the suborder Tetrahymenina . All telomeres consisted of tandemly repeated C4A2 sequences, including the 5' telomere of the 11 kb rDNA from developing macronuclei of Tetrahymena thermophila . Our sequence of the 11 kb 5' telomeric region shows that each one of a previously described pair of inverted repeats flanking the micronuclear rDNA (Yao et al., Mol . Cell . Biol . 5: 1260-1267, 1985) is 29 bp away from the positions to which telomeric C4A2 repeats are joined to the ends of excised 11 kb rDNA . In general we found that the macronuclear rDNA sequences adjacent to C4A2 repeats are not highly conserved . However, in the non-palindromic rDNA of Glaucoma, we identified a single copy of a conserved sequence, repeated in inverted orientation in Tetrahymena spp., which all form palindromic rDNAs . We propose that this sequence is required for a step in rDNA excision common to both Tetrahymena and Glaucoma.

J Cell Sci, 1986 Aug, 84, 237 - 51
Large-scale selection synchrony of Tetrahymena thermophila; Hill RJ et al.; A method is described, based on the phagocytosis of colloidal ferrite particles, which gives highly synchronous populations of Tetrahymena thermophila . To ensure a successful synchrony, the cell culture doubling time, the limits of the phagocytic period and the distribution of cell stages must first be determined . Once these parameters are known, synchrony can be achieved under a variety of growth conditions and with cultures ranging in volume from a few millilitres to 12 litres or more . The main advantages of the method are that the apparatus required is simple, large volumes of cells can be handled easily, and the synchronous populations can be prepared within a few hours . In principle, the method should be applicable to any cell population in which phagocytosis occurs discontinuously over the cell cycle.

Mol Cell Biol, 1986 Aug, 6(8), 3014 - 7
Formation of stable chromatin structures on the histone H4 gene during differentiation in Tetrahymena thermophila; Pederson DS et al.; The relationship between chromatin structure and the transcriptional activity of the histone H4-I gene of Tetrahymena thermophila was explored . Indirect end-labeling studies demonstrated that major DNase I- and micrococcal nuclease-hypersensitive sites flank the active macronuclear genes but not the inactive micronuclear genes . Runon transcription experiments with isolated macronuclei indicated that histone gene transcription rates decreased when cells were starved . However, macronuclear nuclease-hypersensitive sites persisted upon starvation . Thus, one level of transcriptional control of the H4-I gene results in altered chromatin structure and is established during nuclear differentiation . The rate of transcription is also controlled, but not through hypersensitive site-associated structures.

Int J Pept Protein Res, 1986 Aug, 28(2), 179 - 85
Dipeptide synthesis catalyzed by aminoacyl-tRNA synthetases from Bacillus stearothermophilus; Nakajima H et al.; A new approach to enzymatic peptide synthesis by using aminoacyl-tRNA synthetase (ARS) as a catalyst has been investigated . Four ARSs (AspRS, HisRS, LeuRS and TyrRS) have been purified from a thermophilic bacterium, Bacillus stearothermophilus . By using TyrRS as a catalyst, tyrosine and leucinamide were shown to be condensed in the presence of ATP to give tyrosylleucinamide . In this manner, all of the ARSs investigated catalyzed the peptide synthesis reactions . TyrRS did not have strict specificity for the amino acid derivatives used as substrates and even D-amino acids were incorporated into peptides fairly easily in this enzymatic reaction . Preparative scale synthesis of L-Tyr-L-LeuNH2 was carried out and from this the scope and limitation of this new enzymatic reaction as a tool to the peptide synthesis has been described.

Biol Chem Hoppe Seyler, 1986 Aug, 367(8), 723 - 9
Purification, partial characterization and substrate specificity of a squalene cyclase from Bacillus acidocaldarius; Neumann S et al.; From the thermophilic Bacillus acidocaldarius, a membrane bound cyclase catalysing the formation of 22(29)-hopene (diploptene) and 22-hopanol (diplopterol) from squalene was enriched 670-fold to a purity of 95% as judged by gel chromatography . The specific activity of the purified enzyme in the presence of Triton X-100 is 0.22 mumol product formation per min and mg protein at 54 degrees C . The molecular mass is 150 kDa and that of the subunits 80 kDa as determined by FPLC gel chromatography and sodium dodecyl sulfate gel electrophoresis, respectively . Not only squalene but also E,E-homofarnesol, homogeraniol and homofarnesyl (1,5,9-trimethyl-4,8-decadienyl) ether are substrates . The ether bond of the latter substrate is split by the enzyme . The products of the aforementioned three additional substrates are ambroxan (dodecahydro-3a,6,6,9a-tetramethylnaphtho{2,1-b}-furan), octahydro-4,4,7a-trimethylbenzofuran and ambroxan together with 1,5,9-trimethyl-4,8-decadienol . The cyclization rates of these substrates compared to squalene are 3%, 0.05% and 0.3%, respectively . The half-life of the enzyme activity is 80 h at 45 degrees C and 18 h at 54 degrees C.

Dev Biol, 1986 Aug, 116(2), 302 - 13
Developmental regulation of gene expression in Tetrahymena; Mayo KA et al.; The onset of gene expression in Tetrahymena thermophila during macronuclear differentiation was investigated by assay of galactokinase in conjugating deoxygalactose-resistant heterokaryons . Our results distinguish three successive states of galactokinase gene expression for cells developing a new macronucleus: stage 0, refractory to induction; stage 1, inducible by refeeding; and stage 2, induced . The refractory period ends at 12 to 13 hr after the onset of conjugation; this corresponds to the time of pair separation, and occurs several hours after the new macronuclei have become morphologically distinguishable . Stage 1 cells behave indistinguishably from mature starved cells . Inhibitor studies suggest that galactokinase synthesis is induced coincidentally with the induction of bulk protein synthesis during conjugation: thus it behaves developmentally like a typical protein; and that galactokinase mRNA is probably transcribed within 1 hr prior to its translation . Thus, when conjugating cells are refed during the refractory period, some developmental condition prevents the swift induction of protein (and galactokinase) synthesis observed upon refeeding starved (nonmating) cells . The possible nature of this developmental phenomenon is discussed.

Tsitologiia, 1986 Aug, 28(8), 790 - 5
{Stabilizing action of heavy water (D20) on the cell}; Aleksandrov VIa; Literature data and our own findings show convincingly that heavy water (D2O) stabilizing proteins against denaturants and proteinases increases cell thermostability in plants and animals and that of microorganisms . In Drosophila, along with cell and organism thermostabilities, it enhances thermophilia . Evidence is given in favour of the suggestion that the protein stabilization is responsible for the above biological effects of D2O, although heavy water may increase the melting temperature of nucleic acids and, but slightly, of lipids.

J Biol Chem, 1986 Jul 25, 261(21), 9839 - 43
Direct measurement of the electrogenicity of the H+-ATPase from thermophilic bacterium PS3 reconstituted in planar phospholipid bilayers; Hirata H et al.; The proton-translocating ATPase of the thermophilic bacterium PS3 was incorporated into a planar phospholipid bilayer, and its electrogenicity was directly demonstrated . The enzyme (TF0F1) consists of a catalytic portion, F1, and a membrane-integrated portion, Fo . A short-circuit current of up to 1 nA/cm2 was generated upon the addition of ATP, and the direction of the current indicated the flow of positive charges from the TF1 side to the TF0 side . The generation of the electric current was progressively suppressed by the presence of an inhibitor of TF1 such as NaN3 or adenyl-5'-yl imidodiphosphate . An open-circuit membrane potential of 40-120 mV was also demonstrated (more negative on the TF1 side), which was inhibited by NaN3 . Furthermore, an applied voltage of -180 mV (TF1 side negative) was sufficient to prevent the generation of electric current dependent on ATP hydrolysis, which indicated that the electrogenicity of TF0F1 is some 180 mV under the conditions studied . From these results it was tentatively concluded that the number of protons transported across the bilayer/mol of ATP is more than 3.

Biochem J, 1986 Jul 15, 237(2), 415 - 20
The purification and characterization of glucokinase from the thermophile Bacillus stearothermophilus; Goward CR et al.; Homogeneous glucokinase (EC 2.7.1.2) from the thermophile Bacillus stearothermophilus was isolated on the large scale by using four major steps: precipitation of extraneous material at pH 5.5, ion-exchange chromatography on DEAE-Sepharose, pseudo-affinity chromatography on Procion Brown H-3R-Sepharose 4B and gel filtration on Ultrogel AcA 34 . The purified enzyme had a specific activity of about 330 units/mg of protein and was shown to exist as a dimer of subunit Mr 33,000 . Kinetic parameters for the enzyme were determined with a variety of substrates . The glucokinase was highly specific for alpha-D-glucose, and the only other sugar substrate utilized was N-acetyl-alpha-D-glucosamine . The enzyme shows Michaelis-Menten kinetics, with a Km value of 150 microM for alpha-D-glucose . The glucokinase was maximally active at pH 9.0.

Klin Padiatr, 1986 Jul-Aug, 198(4), 344 - 8
{Simultaneous illness in siblings with exogenous allergic alveolitis of the farmer's lung type--report of a 3-year course, familial sensitization and HLA markers}; Rebmann H et al.; Two sisters (6 and 8 years old) fell ill with coughing, fever and dyspnea after playing in the rotten straw in the barn of their parents' farm . Within 2 weeks symptoms changed to the insidious form of Exogen Allergic Alveolitis . In the broad spectrum of antibodies detected, the Thermophile Actinomycetes were probably the relevant antigens . In spite of efforts to avoid contact with the antigens, there was a relapse of the disease, the most probable sources of the antigens being a mattress in the bed-room and a fodderroom . Since consequent avoidance of antigens is practiced, no signs of relapse have been seen . The other three members of the family were exposed too . The mother and the youngest daughter showed neither symptoms nor specific antibodies . The father turned out to be sensibilitized too . By more attentive self-observation he discovered signs of illness . The distribution of the HLA-markers in the family was not concordant with the pattern of disease nor of antibodies . Some HLA-markers have been described as being associated with Exogen Allergic Alveolitis . Among the markers of our family there are none of these.

J Biochem (Tokyo), 1986 Jul, 100(1), 123 - 31
Thermophilic HB8 DNA ligase: effects of polyethylene glycols and polyamines on blunt-end ligation of DNA; Takahashi M et al.; NB8 DNA ligase from an extract of Thermus thermophilus HB8 could catalyze blunt-end ligation in the presence of high concentration of polyethylene glycols (PEG) or in the presence of polyamines . In the presence of high molecular weight PEG 20,000, 6,000, or 1,000 (8-28%), the enzyme catalyzed blunt-end intermolecular joining to yield linear oligomers, but no circular DNA forms . But in the presence of low molecular PEG 400, 200 (8-80%), or the monomer, ethylene glycol (16-80%), the circular forms were also detected by intramolecular ligation . In the presence of polyamines, the blunt-end ligation products were linear oligomers and the optimum concentrations were as follows: caldopentamine (0.05 mM), thermine (0.1-0.2 mM), spermine (0.2 mM), thermospermine (0.4 mM), and sperminediol (0.75 mM) . Spermidine and putrescine were less capable of producing oligomers . PEG and polyamines elevated the ligation temperature by HB8 DNA ligase . The optimum temperature of blunt-end ligation was about 65 degrees C.

Mol Cell Biol, 1986 Jul, 6(7), 2527 - 35
Effect of heat shock on ribosome structure: appearance of a new ribosome-associated protein; McMullin TW et al.; After a nonlethal but heat shock protein-inducing hyperthermic treatment, ribosomes isolated from Tetrahymena thermophila contained an additional 22-kilodalton protein (p22) . When maximally ribosome associated, this protein was found to be on the small subunit in a 1:1 stoichiometric ratio with other ribosomal proteins . Using an antiserum directed against the purified 22-kilodalton protein, we found that non-heat-shocked and heat-shocked cells contain identical amounts of this protein, the only difference being that in the stressed cells p22 is entirely ribosome bound, whereas in the unstressed cells p22 has little or no detectable ribosome association . Because the two-dimensional electrophoretic properties of p22 showed no alterations after heat shock, this change in state of ribosome-p22 interaction does not appear to be caused by a chemical modification of p22 . When not strongly ribosome associated, p22 is not found free in the cytoplasm . During that time in heat shock when p22 is first becoming ribosome associated, it is found preferentially on polysomal ribosomes . Subsequently, all ribosomes, whether polysome bound or not, obtain a bound p22 . The functional significance of this association is discussed.

Mol Biol Evol, 1986 Jul, 3(4), 343 - 55
The simple repeat poly(dT-dG).poly(dC-dA) common to eukaryotes is absent from eubacteria and archaebacteria and rare in protozoans; Morris J et al.; Genomic DNA from a wide variety of prokaryotic and eukaryotic organisms has been assayed for the simple repeat sequence poly(dT-dG).poly(dC-dA) by Southern blotting and DNA slot blot hybridizations . Consistent with findings of others, we have found the simple alternating sequence to be present in multiple copies in all organisms in the animal kingdom (e.g., mammals, reptiles, amphibians, fish, crustaceans, insects, jellyfish, nematodes) . The TG element was also found in lower eukaryotes (Saccharomyces cerevisiae, Neurospora crassa, and Dictyostelium discoideum) and at a much lower frequency in protozoans (Oxytricha fallux and Tetrahymena thermophila) . The sequence was also repeated in high copy number in a higher plant (Zea mays) as well as at very high levels in a unicellular green alga (Chlamydomonas reinhardi) . Although the copy number of the repeat per haploid genome was generally proportional to genome size, there was a greater-than-1,000-fold variation in the number of (TG)25/100-kb genomic DNA . By contrast, no eu-or archaebacterium--including Myxococcus xanthus, whose life cycle is very similar to that of the slime mold Dictyostelium discoideum, and Halobacter volcanii, whose genome contains other repeated sequences--was found whose genomic DNA contained this sequence in detectable amounts . A computer search also failed to find the TG element in human mitochondrial DNA.

Eur J Biochem, 1986 Jul 1, 158(1), 77 - 83
Purification and characterization of Bacillus coagulans oligo-1,6-glucosidase; Suzuki Y et al.; A p-nitrophenyl-alpha-D-glucopyranoside-hydrolyzing oligo-1,6-glucosidase of Bacillus coagulans ATCC 7050 (facultative thermophile) was purified to homogeneity . The relative molecular mass, Stokes radius, sedimentation coefficient at 20 degrees C in water, molecular absorption coefficient at 280 nm and pH 6.8, and isoelectric point were estimated as 60 000, 3.29 nm, 4.8 X 10(-13) s, 1.34 X 10(5) M-1 cm-1, and 4.3, respectively . The amino-terminal amino acid was threonine . There was no common antigenic group between the enzyme and each of its homologous counterparts from Bacillus cereus ATCC 7064 (mesophile) and Bacillus thermoglucosidasius KP 1006 (obligate thermophile) . These oligo-1,6-glucosidases strongly resembled one another in their amino acid composition, except that the proline content increased with the elevation of thermostability in the order, mesophile----facultative thermophile----obligate thermophile enzymes.

Mol Cell Biol, 1986 Jul, 6(7), 2364 - 70
Site-specific methylation of adenine in the nuclear genome of a eucaryote, Tetrahymena thermophila; Harrison GS et al.; DNA in the polyploid macronucleus of the ciliated protozoan Tetrahymena thermophila contains the modified base N6-methyladenine . We identified two GATC sites which are methylated in most or all of the 45 copies of the macronuclear genome . One site is 2 kilobases 5' to the histone H4-I gene, and the other is 5 kilobases 3' to the 73-kilodalton heat shock protein gene . These sites are de novo methylated between 10 and 16 h after initiation of conjugation, during macronuclear anlage development . The methylation states of these two GATC sites and four other unmethylated GATC sites do not change in the DNA of cells cultured under conditions which change the activity of the genes, including logarithmic growth, starvation, and heat shock.

EMBO J, 1986 Jul, 5(7), 1515 - 9
Evidence for dimer structure of proton-pumping cytochrome c oxidase, an analysis by radiation inactivation; Sone N et al.; Cytochrome c oxidases, purified from bovine heart and the thermophilic bacterium PS3, were irradiated with a high-energy electron beam . The proton transport activities of both preparations and their electron transfer activities decreased as single exponential functions of the radiation dosage . Applying the target theory with alkaline phosphatase as an internal standard, the following functional molecular weights were obtained for cytochrome c oxidation and H+ pumping: 63-73 kd and 160-220 kd, respectively, for the bovine enzyme, and 80-100 kd and 190-230 kd for the PS3 enzyme . The results suggest that a dimer structure is necessary for H+ pumping, while a core part of monomer (presumably the largest two subunits, i.e . subunits I and II) is sufficient for cytochrome c oxidation.

Appl Environ Microbiol, 1986 Jul, 52(1), 45 - 50
Cloning and expression of the beta-D-galactosidase gene from Streptococcus thermophilus in Escherichia coli; Herman RE et al.; The beta-D-galactosidase (beta-gal) gene from Streptococcus thermophilus was cloned to isolate and characterize it for potential use as a selection marker in a food-grade cloning vector . Chromosomal DNA from S . thermophilus 19258 was cleaved with the restriction enzyme PstI and ligated to pBR322 for transformation into Escherichia coli JM108 . A beta-galactosidase-positive clone was detected by its blue color on a medium supplemented with 5-bromo-4-chloro-3-indolyl-beta-D-galactoside . This transformant possessed a single plasmid, designated pRH116, which contained, in addition to the vector DNA, a 7.0-kilobase (kb) PstI insertion fragment coding for beta-gal activity . An extract from JM108(pRH116) contained a beta-gal protein with the same electrophoretic mobility as the beta-gal from S . thermophilus 19258 . Compared with the beta-gal from E . coli HB101, the S . thermophilus beta-gal was of lower molecular weight . A restriction map of pRH116 was constructed from cleavage of both the plasmid and the purified insert . The construction of deletion derivatives of pRH116 with BglII, BstEII, and HindIII revealed the approximate location of the gene on the 7.0-kb fragment . The beta-gal gene was further localized to a 3.85-kb region.

Biochim Biophys Acta, 1986 Jun 20, 867(3), 97 - 106
Stability of structures of the epsilon subunit and terminator of thermophilic ATPase; Saishu T et al.; F1-type ATPase is the central enzyme for ATP synthesis in most organisms . Because of the extreme reconstitutability of thermophilic ATPase (TF1) and diversity of the minor subunits of F1 type ATPase, an operon coding for TF1 was isolated from DNA of thermophilic bacterium PS3, and its terminal region containing the epsilon subunit (TF1 epsilon) and terminator was sequenced . The primary structure of the epsilon subunit (Mr = 14 333) was deduced from the nucleotide sequence (396 base-pairs) and amino-acid sequence of its amino terminus . The conclusions drawn from the results are as follows . Homologies: TF1 epsilon shows only 6% homology with the epsilon subunits of eight species reported, but 50% homology with Escherichia coli epsilon and 41% with chloroplast . The residues having a tendency to form reverse turns (Gly, Pro and Tyr) and His are relatively well conserved . Unlike some F1 epsilon types TF1 epsilon has no ATPase inhibitor activity and is not homologous with ATPase inhibitor . TF1 epsilon is essential to connect F1 to F0, like the b subunit, and is weakly homologous with the b subunit of F0F1 . The cause of 3 beta: 1 epsilon subunit stoichiometry: The ribosome binding sequence of TF1 epsilon is TAGGN7, which is incomplete compared with that of TF1 beta . The codon usage for TF1 epsilon is similar to that for TF1 epsilon . The cause of stability of TF1 epsilon and its gene: There are 18 ionic groups at the putative reverse turns and the N- and C-termini of TF1 epsilon, but only 10 ionic groups in the corresponding sites of E . coli epsilon subunit . These ionic groups enhance the external polarity of TF1 epsilon and may intensify subunit-subunit interaction . There is a terminator at the 3' end of the TF1 epsilon gene, which is stabilized by a long (13 base-pairs) stem.

Eur J Biochem, 1986 Jun 16, 157(3), 455 - 62
Differential features of ribosomes and of poly(U)-programmed cell-free systems derived from sulphur-dependent archaebacterial species; Londei P et al.; The properties of poly(U)-directed cell-free systems developed from the sulphur-dependent, thermophilic archaebacteria Desulfurococcus mobilis, Thermoproteus tenax, Sulfolobus solfataricus, Thermococcus celer and Thermoplasma acidophilum have been compared . All systems are truly thermophilic in requiring incubation at temperatures close to the physiological optimum for cell growth . Under optimized conditions the error frequency in tRNA selection is less than 0.4% at 80 degrees C, and synthetic efficiencies (Phe residues polymerized per ribosome in 40 min) span from 4 for Tp . tenax, to 10 for Tc . celer, to 20-25 for D . mobilis and T . acidophilum and to 40 for S . solfataricus . According to requirements for polypeptide synthesis and to degree of stability of the ribosomal subunits' association, sulphur-dependent thermophiles cluster into two groups . Group I organisms (D . mobilis, Tp . tenax, S . solfataricus) harbour 70-S monomers composed of weakly associated subunits, whose poly(Phe)-synthesizing capacity is totally dependent on added spermine while being drastically inhibited by monovalent cations . Group II organisms (Tc . celer and T . acidophilum) contain 70-S particles composed of tightly bonded subunits, whose synthetic capacity is independent of spermine while being totally dependent on monovalent cations . Spermine promotes poly(Phe) synthesis on ribosomes of group I organisms by converting the peptidyltransferase center into an active conformation, while monovalent cations are inhibitory by preventing the interaction between the free ribosomal subunits . The closeness between Tc . celer and T . acidophilum ribosomes provides new insight on the phylogenetic placement of Thermococcaceae.

Biochim Biophys Acta, 1986 Jun 10, 850(1), 139 - 45
Iron-histidine stretching Raman line and enzymic activities of bovine and bacterial cytochrome c oxidases; Sone N et al.; Resonance Raman spectra of the reduced form of cytochrome c oxidase isolated from bovine heart and the thermophilic bacterium PS3 were investigated in relation to their H+-pumping- and cytochrome-c-oxidizing activities, which were varied by incubating the enzyme at raised temperatures or at alkaline pH at room temperature . For both the bovine and PS3 enzymes, the intensity of the iron-histidine stretching Raman line of the ferrous a3 heme (214 cm-1) exhibited an incubation-temperature-dependent change, which fell between the similar curves of the H+-pumping and cytochrome-c-oxidizing activities . The intensities of the formyl CH=O stretching Raman line of the ferrous a3 heme (1665 cm-1) as well as of other lines were insensitive to the heat treatment . The iron-histidine stretching Raman line of both enzymes showed pH-dependent intensity change which was nearly parallel with the pH dependence of cytochrome-c-oxidizing activity . Therefore, deprotonation affecting the 214 cm-1 Raman line is responsible for the decrease of activity . This limited alkaline treatment to the PS3 enzyme was reversible and the recovered enzyme exhibited Raman intensities and enzymic activities similar to the native one . However, the neutralized, bovine enzyme with a similar intensity of the 214 cm-1 line showed increased cytochrome-c-oxidizing activity and null H+-pumping activity.

J Biol Chem, 1986 Jun 5, 261(16), 7566 - 70
Protein secretion in Tetrahymena thermophila . Characterization of the major proteinaceous secretory proteins; Maihle NJ et al.; The contents of mucocysts of the ciliated protozoan Tetrahymena thermophila comprise about 12 proteins, ranging in relative mobility (Mr) from approximately 160,000 to 8,000 . There are at least four families of sulfhydryl-linked mucocyst polypeptides . One of these families includes a prominent Mr 34,000 protein, as determined by one- and two-dimensional gel electrophoresis . The Mr 34,000 protein is resolved into two species in isoelectric focusing gels, with apparent pI values of 4.8 and 4.9; most of the other mucocyst proteins also exhibit acidic apparent isoelectric points . The identity of the major Mr 34,000 protein as a bona fide mucocyst component is substantiated by indirect immunofluorescent localization of this protein in a linear punctate pattern coincident with the localization of mucocysts in these cells; this pattern of localization can be abolished by stimulation of synchronous secretion and is absent in a mutant strain devoid of these secretory organelles (Maihle, N . J., and Satir, B . H . (1985a) J . Cell Sci . 78, 49-65.

Biochemistry, 1986 Jun 3, 25(11), 3275 - 82
Time-dependent inhibition of Bacillus stearothermophilus alanine racemase by (1-aminoethyl)phosphonate isomers by isomerization to noncovalent slowly dissociating enzyme-(1-aminoethyl)phosphonate complexes; Badet B et al.; An alanine racemase encoded by a gene from the thermophilic Gram-positive bacterium Bacillus stearothermophilus is overproduced to 0.3% of the soluble protein when carried on plasmid pICR4 in Escherichia coli {Inagaki, K., Tanizawa, K., Badet, B., Walsh, C . T., Tanaka, H., & Soda, K . (1986) Biochemistry (third paper of four in this issue)} . Purification of large quantities (50 mg) of racemase permits study of time-dependent inactivation by D and L isomers of the antibacterial (1-aminoethyl)phosphonate (Ala-P), the phosphonate analogue of alanine . The time-dependent activity loss by this compound now appears general to Gram-positive but not to Gram-negative racemases {Badet, B., & Walsh, C . (1985) Biochemistry 24, 1333} and is shown to occur by extremely slow dissociation of a noncovalent E X Ala-P complex . Ala-P binds initially in a weak, reversible (KI = 1 mM) competitive manner but is slowly isomerized (kinact = 6-9 min-1) to a stoichiometric enzyme complex, which in turn dissociates extremely slowly, with a half-time about 25 days . Thus, Ala-P is a slow but not a tight-binding inhibitor . The E X Ala-P complex is not reducible by borohydride but does perturb the fluorescence of bound pyridoxal 5'-phosphate coenzyme . Determination of the sequence of an active site octapeptide of the B . stearothermophilus alanine racemase shows homology with the sequence of a Gram-negative Salmonella typhimurium alanine racemase that is not susceptible to time-dependent inhibition by Ala-P . Studies with Ala-P analogues suggest the phosphonate dianion is crucial for stable formation of an isomerized long-lived E X Ala-P-inhibited complex.

Biochemistry, 1986 Jun 3, 25(11), 3268 - 74
Thermostable alanine racemase from Bacillus stearothermophilus: molecular cloning of the gene, enzyme purification, and characterization; Inagaki K et al.; The alanine racemase (EC 5.1.1.1) gene of a thermophilic bacterium, Bacillus stearothermophilus, was cloned and expressed in Escherichia coli C600 with vector plasmid pICR301, which was constructed from pBR322 and the L-alanine dehydrogenase gene derived from B . stearothermophilus . A coupled assay method with L-alanine dehydrogenase and tetrazolium salts was used to detect visually the alanine racemase activity in the clones . Alanine racemase overproduced in a clone carrying the plasmid pICR4, 12 kilobases of DNA, was purified from cell extracts about 340-fold to homogeneity by five steps including heat treatment . The overproduced enzyme was confirmed to originate from B . stearothermophilus by an immunochemical cross-reaction with the enzyme of B . stearothermophilus . The purified enzyme has a molecular weight of about 78 000 and consists of two identical subunits of Mr of 39 000 . At the optimum temperature (50 degrees C), the enzyme has a specific activity of 1800 units/mg (Vmax, D- to L-alanine) . Resolution and reconstitution experiments together with the absorption spectrum of the enzyme clearly indicate that alanine racemase of B . stearothermophilus is a pyridoxal 5'-phosphate enzyme.

J Gen Microbiol, 1986 Jun, 132 ( Pt 6), 1709 - 22
The isolation and characterization of bacteriophages infecting obligately thermophilic strains of Bacillus; Sharp RJ et al.; Twenty-four thermophilic bacteriophages have been isolated from diverse sources such as compost, soil, silage and rotting straw . Although considerable individual host specificity was observed, the phages were able to infect most of the major taxonomic groups of Bacillus thermophiles . The phages varied considerably in morphology and size; the phage heads were either cylindrical or polyhedral with tails varying in length between 15 and 500 nm . Most of the phages were stable at 50 degrees C for 4-5 h but at 70 degrees C the plaque-forming units decreased by between 10(2)- and 10(7)-fold in 2 h . The DNA of morphologically similar phages was examined by restriction enzyme analysis, and some differences in the DNA fragment patterns were found . Efficiency of plating data indicated that 'B . caldotenax' has a restriction and modification system . These phages may be valuable for the study of the genetics of thermophilic bacilli: transduction of 'B . caldotenax' and 'B . caldovelox' by phage JS017 has been observed.

J Gen Microbiol, 1986 Jun, 132 ( Pt 6), 1677 - 83
Isolation and preliminary taxonomic studies of Thermus strains isolated from Yellowstone National Park, USA; Munster MJ et al.; Forty-eight strains of Thermus isolated from hot springs in Yellowstone National Park, Wyoming, USA, and eight reference strains were subjected to a numerical taxonomic analysis using Gower's coefficient (SG) with single and average linkage clustering . Two major groups were distinguished, which could be differentiated by colony morphology, ability to reduce nitrate and proteolytic activity . Cluster 1 contained Thermus aquaticus YT-1, the type strain of the species, and cluster 2 contained authentic strains of 'T . flavus' and 'T . thermophilus' . T . ruber was recovered as a single member cluster . The mol % G + C of DNA from representative strains from each cluster was 64.4 to 66.8 for cluster 1, 62.2 to 67.1 for cluster 2 and 62.5 for T . ruber.

J Cell Sci, 1986 Jun, 82, 223 - 34
Homotypic pair formation during conjugation in Tetrahymena thermophila; Kitamura A et al.; In the ciliate Tetrahymena thermophila, conjugation has been believed to occur only between cells of different mating types . We found the formation of homotypic pairs during normal conjugation by using micronuclear morphological markers . Homotypic pairs formed preferentially during the first 10 min following the first pair formation and comprised about half of the pairs . These results suggest the involvement of mating-type non-specific adhesion of cells in the initial step of conjugation . Homotypic pairs apparently persist for at least 30 min and then separate into single cells . Homotypic pairs are also formed when conjugant pairs re-form after mechanical separation of heterotypic pairs . Five kinds of glycosidases, three kinds of proteases and phospholipase C showed no effect on either the formation of homotypic pairs of their separation . The relation between the mating-type substances and the molecules responsible for mating-type non-specific adhesion of cells is discussed.

Mol Cell Biol, 1986 Jun, 6(6), 2267 - 70
No heat shock protein synthesis is required for induced thermostabilization of translational machinery; Hallberg RL; For Tetrahymena thermophila cells to survive at 43 degrees C, a normally lethal temperature, they require a pretreatment which either elicits the synthesis of heat shock proteins or one which brings about a change in the translational machinery of the cell such that is is not inactivated when transferred to 43 degrees C . In this report I present evidence showing that the latter modification can occur in the complete absence of protein synthesis, indicating that heat shock protein production is not required for the induced thermostabilization of the translational machinery.

J Biochem (Tokyo), 1986 Jun, 99(6), 1667 - 72
Characterization of an essential histidine residue in thermophilic malate dehydrogenase; Iijima S et al.; Heat-stable malate dehydrogenase isolated from Thermus flavus AT62 was completely inactivated by treatment with diethylpyrocarbonate . The inactivation was accompanied by the loss of 1.2 histidine residues per subunit of the enzyme . The enzyme was protected from inactivation by NADH . The enzyme was also inactivated by dye-sensitized photooxidation . Methionine residues, in addition to histidine residues, were destroyed in the inactivated enzyme . Kinetic analyses of the inactivation indicated that the pK value of the residue involved in the inactivation was 8.20 at 25.0 degrees C and 7.52 at 60.0 degrees C . From the pK values and the heat of ionization calculated from the van't Hoff plot of pKs, a histidine residue was identified to be primarily involved in the inactivation . The effect of temperature on the pK value of the essential group in this enzyme from a thermophilic organism is discussed.

Cell Differ, 1986 Jun, 18(4), 243 - 56
Developmental and genetic effects of alcian blue in conjugating Tetrahymena thermophila: doublet formation and macronuclear retention; Gutierrez JC et al.; Genetic, kinetical and cortical effects of treatment with the inducer of mucocyst release, alcian blue (AB), on conjugating pairs of Tetrahymena thermophila are reported . AB induces the formation of doublet cells from pairs, and the majority of them are homopolar doublets . We present a model in order to explain the origin of these cells . Macronuclear retention (MR) is the most important genetic effect observed . Two kinds of MR can be obtained: prezygotic-MR (uniparental micronucleus) and postzygotic-MR (cross-fertilized micronucleus) . Within the first group, both homokaryon and heterokaryon cells are obtained . From some abnormal conjugational configurations and the results of conjugational kinetic analysis we propose an explanation for the origin of MR cells induced by AB . Genetic effects obtained after AB treatment at different conjugational times are independent of the cortical ones . The utility of these different effects in genetical and physiological studies is discussed.

Exp Cell Res, 1986 Jun, 164(2), 562 - 7
Post-meiotic DNA synthesis in nocodazole-blocked nuclei during conjugation of Tetrahymena thermophila . Induction of polyploidy in the micronucleus; Gaertig J et al.; The formation of single, polyploid micronuclei was induced during conjugation of Tetrahymena thermophila with Nocodazole (ND) according to Kaczanowski et al . The increase in DNA content in these nuclei was measured cytophotometrically in conjugating pairs continuously exposed to the drug . ND-treated micronuclei ('restitution nuclei') undergo two complete rounds of DNA replication and enter the third at the time of pair separation . The DNA content of these micronuclei in late pairs was in the range 16-30C (mean 20C) . This amount was similar to the sum of the DNA content of all post-meiotic products during normal conjugation at about the same stage . Thus the increase in DNA content in 'restitution' nuclei reflects some intrinsic ability of nuclei in pairing cells to replicate DNA independently of nuclear division.

Proc Natl Acad Sci U S A, 1986 Jun, 83(12), 4369 - 73
Transformation of Tetrahymena thermophila by microinjection of ribosomal RNA genes; Tondravi MM et al.; The ribosomal RNA genes (rDNA) of Tetrahymena thermophila macronucleus exist as free linear 21-kilobase molecules that contain replication origins and telomeres . A mutation in this gene confers resistance to the antibiotic paromomycin . We have isolated rDNA from such a mutant (strain p2f), microinjected it into the macronucleus of a sensitive strain, and obtained drug-resistant cells at a frequency of 1-3% . The transformed cells have a distinct and stable phenotype . The rDNA of the transformants contains the expected sequences of the mutant rDNA as determined by oligonucleotide hybridization . rDNA from a different inbred line (C3-368), which contains heteromorphic restriction sites, has also been used for injection, and the results confirm the fact that the injected rDNA is indeed present in the transformants . Injection of rDNA from the C3 strains also increases the transformation frequency 5- to 10-fold and leads to the total replacement of the resident rDNA of the B-inbred strains . This is presumably due to the replication dominance of rDNA from the C3 strains over that of the B strains . Using this method, we have also been able to transform developing cells, at similar frequencies, by microinjecting into the macronuclear anlagen.

Biol Chem Hoppe Seyler, 1986 Jun, 367(6), 473 - 81
DNA-dependent RNA polymerases of the three orders of methanogens; Thomm M et al.; The DNA-dependent RNA polymerases of members of the three orders of methanogens were purified and their enzymatic properties described . The enzymes consist of 7-8 polypeptides . Although these differed in molecular mass, the four heaviest components could be allied to components of the enzyme of Methanobacterium thermoautotrophicum, W by cross-reaction with antibodies directed against the denatured polypeptides of this enzyme . The antisera against native RNA polymerases isolated from representatives of the different orders, on the other hand, gave rise to serological cross-reaction between different genera but not between different families and orders . These antisera are thus useful for taxonomic purposes . The RNA polymerase of the extreme thermophile Methanothermus fervidus shows a rather low thermostability . No factors having a stabilizing influence on the enzyme could be detected.

J Bacteriol, 1986 Jun, 166(3), 1046 - 54
Ultrastructure of the cell envelope of the archaebacteria Thermoproteus tenax and Thermoproteus neutrophilus; Messner P et al.; The ultrastructures of the regular surface layers (S-layers) of the extremely thermophilic archaebacteria Thermoproteus tenax and Thermoproteus neutrophilus were examined by freeze-etching, freeze-drying, and negative staining methods combined with optical and digital image enhancement . In both strains, a monolayer of macromolecules arranged in hexagonal arrays with center-to-center spacings of approximately 30 nm was the only component of the cell wall . The gross morphologies of the S-layer lattices of the two organisms were similar and showed the same handedness in the arrangement of the protomers of the morphological units . Striking differences were found in the anionic charge distributions on the surfaces of the two S-layer proteins as determined by labeling with polycationic ferritin . Analysis of the lattice orientation, together with the number and distribution of lattice faults on intact cells, provided a strong indication that the S-layers of both organisms have a shape-determining function.

Proc Natl Acad Sci U S A, 1986 Jun, 83(12), 4360 - 3
A model for the RNA-catalyzed replication of RNA; Cech TR; A shortened form of the self-splicing ribosomal RNA intervening sequence of Tetrahymena thermophila has enzymatic activity as a poly(cytidylic acid) polymerase {Zaug, A.J . & Cech, T.R . (1986) Science 231, 470-475} . Based on the known properties of this enzyme, a detailed model is developed for the template-dependent synthesis of RNA by an RNA polymerase itself made of RNA . The monomer units for RNA synthesis are tetra- and pentanucleotides of random base sequence . Polymerization occurs in a 5'-to-3' direction, and elongation rates are expected to approach two residues per minute . If the RNA enzyme could use another copy of itself as a template, RNA self-replication could be achieved . Thus, it seems possible that RNA catalysts might have played a part in prebiotic nucleic acid replication, prior to the availability of useful proteins.

Biochem Biophys Res Commun, 1986 May 14, 136(3), 899 - 905
The effect of anionic detergents on the ATPase activity of isolated F1 from the thermophilic bacterium PS3; Norling B; The ATPase activity of F1 isolated from the thermophilic bacterium PS3 is stimulated at 30 degrees C by the anionic detergents cholate or deoxycholate . Maximal activity obtained with these detergents (35 mumol/min X mg) is similar to the activity reported for the optimal temperature, 75 degrees C . The activity is linearly stimulated by the detergents and maximal activity is obtained at the critical micellar concentration of the respective detergent . The results are discussed in relation to the role of subunit interactions of the oligomeric enzyme during catalysis and the mode of interaction between the subunits.

J Biol Chem, 1986 May 5, 261(13), 5714 - 21
Characterization of the catalytic and noncatalytic ADP binding sites of the F1-ATPase from the thermophilic bacterium, PS3; Yoshida M et al.; Two classes of ADP binding sites at 20 degrees C have been characterized in the F1-ATPase from the thermophilic bacterium, PS3 (TF1) . One class is comprised of three sites which saturate with {3H}ADP in less than 10 s with a Kd of 10 microM which, once filled, exchange rapidly with medium ADP . The binding of ADP to these sites is dependent on Mg2+ . {3H}ADP bound to these sites is removed by repeated gel filtrations on centrifuge columns equilibrated with ADP free medium . The other class is comprised of a single site which saturates with {3H}ADP in 30 min with a Kd of 30 microM . {3H}ADP bound to this site does not exchange with medium ADP nor does it dissociate on gel filtration through centrifuge columns equilibrated with ADP free medium . Binding of {3H}ADP to this site is weaker in the presence of Mg2+ where the Kd for ADP is about 100 microM . {3H}ADP dissociated from this site when ATP plus Mg2+ was added to the complex while it remained bound in the presence of ATP alone or in the presence of ADP, Pi, or ADP plus Pi with or without added Mg2+ . Significant amounts of ADP in the 1:1 TF1.ADP complex were converted to ATP in the presence of Pi, Mg2+, and 50% dimethyl sulfoxide . Enzyme-bound ATP synthesis was abolished by chemical modification of a specific glutamic acid residue by dicyclohexylcarbodiimide, but not by modification of a specific tyrosine residue with 7-chloro-4-nitrobenzofurazan . Difference circular dichroism spectra revealed that the three Mg2+ -dependent, high affinity ADP binding sites that were not stable to gel filtration were on the alpha subunits and that the single ADP binding site that was stable to gel filtration was on one of the three beta subunits . It has also been demonstrated that enzyme-bound ATP is formed when the TF0.F1 complex containing bound ADP was incubated with Pi, Mg2+, and 50% dimethyl sulfoxide.

J Microsc, 1986 May, 142 ( Pt 2), 247 - 58
Electron microscopic study of 5S rRNA crystals from Thermus thermophilus HB8; Morikawa K et al.; Several types of crystals were grown from 5S rRNA, which was purified from the highly thermophilic bacterium, Thermus thermophilus HB8 . Crystal lattice parameters were determined by X-ray and electron diffraction . One type of crystal was suitable for electron microscopy after staining with uranyl acetate . Two projections derived from micrographs taken at different tilt angles were processed for image analysis . This result enabled us to deduce the arrangement of molecules within the crystal lattice.

J Protozool, 1986 May, 33(2), 216 - 8
Kinetics of the DFPase activity in Tetrahymena thermophila; Landis WG et al.; Crude homogenates of the ciliate protozoon, Tetrahymena thermophila, can hydrolyze the potent acetylcholinesterase inhibitors O,O-diisopropylphosphorofluoridate (DFP) and O-1,2,2-trimethylpropylmethylphosphonofluoride (soman) . Characterization of the enzymatic activity of the homogenate has been performed . The DFPase operates over a pH range of 4 to 10 and an ionic range of 0-500 mM NaCl . Rate of reaction increases three- to four-fold from 25 degrees C to 40 degrees C and is still present at 55 degrees C . These results indicate that the enzymatic activity operates over a broad range of environmental conditions, making it an attractive material for use in the detoxification and detection of organofluorophosphates . DFPases may be important in the metabolism of naturally occurring organophosphates.

J Protozool, 1986 May, 33(2), 204 - 8
Purification and partial characterization of the H immobilization antigens of Tetrahymena thermophila; Doerder FP et al.; The H immobilization antigens specified by the SerH locus of Tetrahymena thermophila have been purified by a procedure utilizing acid fractionation, ammonium sulfate precipitation, gel filtration, and ion exchange chromatography . Purified antigen migrates as a single band on SDS-PAGE and IEF . Molecular weights of the four allelic H antigens range from 44,000 to 52,000, and isoelectric points range from 4.1 to 4.5 . No carbohydrate was detected.

J Hered, 1986 May-Jun, 77(3), 202 - 4
Dominant mutations regulating i-antigen expression in Tetrahymena thermophila; Doerder FP; Two dominant mutations at the RseD locus regulating the differential expression of alternative cell surface immobilization antigens of the ciliate Tetrahymena thermophila are described . RseD1 and RseD2 express I to the exclusion of H (28 degrees C) and are leaky for I when expressing either L (15 degrees C) or T (40 degrees C) . Complementation was not observed in RseD1/RseD2 heterozygotes, and in 326 testcross progeny no wild-type (micronuclear) recombinants were observed . Macronuclear recombination also was not observed . RseD is located on chromosome 5, at least 50 map units from rseB, which also regulates antigen expression . This brings to four the number of loci known to regulate antigen expression.

Zentralbl Bakteriol Mikrobiol Hyg {B}, 1986 May, 182(3), 241 - 53
{Disinfection of sewage sludge by aerobic thermophilic treatment}; Filip Z et al.; Thermal stabilization of sewage sludge, performed in a pilot plant under aerobic conditions, resulted in microbiological and virological decontamination . A minimum temperature of 50 degrees C was necessary to inactivate coliform bacteria, E . coli and Salmonella sp . The rate of inactivation of these bacteria and enteric viruses was dependent on the concentration of microbes in the raw sludge, the temperature and the detention time of the sludge . At a temperature of 56 +/- 1 degree C, which was achieved by autogeneous heating of the processed sludge, pathogens were usually inactivated after two hours, but the time required for inactivation was shorter when the temperature was higher.

J Bacteriol, 1986 May, 166(2), 635 - 43
Structural genes encoding the thermophilic alpha-amylases of Bacillus stearothermophilus and Bacillus licheniformis; Gray GL et al.; The genes encoding the thermostable alpha-amylases of Bacillus stearothermophilus and B . licheniformis were cloned in Escherichia coli, and their DNA sequences were determined . The coding and deduced polypeptide sequences are 59 and 62% homologous to each other, respectively . The B . stearothermophilus protein differs most significantly from that of B . licheniformis in that it possesses a 32-residue COOH-terminal tail . Transformation of E . coli with vectors containing either gene resulted in the synthesis and secretion of active enzymes similar to those produced by the parental organisms . A plasmid was constructed in which the promoter and the NH2-terminal two-thirds of the B . stearothermophilus coding sequence was fused out of frame to the entire mature coding sequence of the B . licheniformis gene . Approximately 1 in 5,000 colonies transformed with this plasmid was found to secrete an active amylase . Hybridization analysis of plasmids isolated from these amylase-positive colonies indicated that the parental coding sequences had recombined by homologous recombination . DNA sequence analysis of selected hybrid genes revealed symmetrical, nonrandom distribution of loci at which the crossovers had resolved . Several purified hybrid alpha-amylases were characterized and found to differ with respect to thermostability and specific activity.

Biochemistry, 1986 Apr 22, 25(8), 1887 - 91
Natural variation of tyrosyl-tRNA synthetase and comparison with engineered mutants; Jones MD et al.; We report the cloning and sequence analysis of the gene for the tyrosyl-tRNA synthetase from Bacillus caldotenax and properties of the gene product . The amino acid sequence of the tyrosyl-tRNA synthetase was found to be 99% homologous with the corresponding enzyme from B . stearothermophilus, with only four amino acid differences . Two of these natural variations were found to involve active site residues of the enzyme and correspond to mutations that have been engineered previously in vitro . One, Thr-51----Ala-51, produced a more active enzyme, possessing a higher value of kcat/KM for ATP . Position 51 is a "hot spot" in the tyrosyl-tRNA synthetase, differing in enzymes derived from Escherichia coli, B . stearothermophilus, and B . caldotenax . The other, His-48----Asn-48, is found to be a neutral mutation but is in one of the rare regions that are conserved with other aminoacyl-tRNA synthetases . The equivalence of histidine and asparagine at position 48 extends the homology in this region to more enzymes . These residues, His-Ile-Gly-His, and now His-Ile-Gly-Asn, form part of the binding site for ATP in the transition state of the reaction . Although B . caldotenax is an obligate thermophile with an optimal growth temperature of 80 degrees C, as much as 20 degrees C above the growth optima of strains of Bacillus stearothermophilus, its tyrosyl-tRNA synthetase has an identical thermal stability in vitro to that from B . stearothermophilus.

Biochemistry, 1986 Apr 8, 25(7), 1529 - 34
NMR study of isoleucine transfer RNA from Thermus thermophilus; Choi BS et al.; An NMR and nuclear Overhauser effect (NOE) analysis of Thermus thermophilus tRNAIle1a is presented . This species contains modifications including s2T54 and s4U8 {Horie, N., Hara-Yokoyama, M., Yokoyama, S., Watanabe, K., Kuchino, Y., Nishimura, S., & Miyazawa, T . (1985) Biochemistry 24, 5711-5715} . All the expected secondary and reverse Hoogsteen AU pairs were identified, with one possible exception . The general geometry of the T psi C loop is the same as the Escherichia coli species, and there is NOE evidence for an A9-UA12 triple . Preliminary measurements of solvent exchange rates of internally hydrogen-bonded bases suggest that this tRNA is more stable than previously studied E . coli and yeast tRNAs.

J Bacteriol, 1986 Apr, 166(1), 338 - 40
Genetic transformation of the extreme thermophile Thermus thermophilus and of other Thermus spp; Koyama Y et al.; Genetic transformation of auxotrophs of the extreme thermophile Thermus thermophilus HB27 to prototrophy was obtained at high frequencies of 10(-2) to 10(-1) when proliferating cell populations were exposed to chromosomal DNA from a nutritionally independent wild-type strain . The transformation frequency was proportional to the DNA concentration from 10 pg/ml to 100 ng/ml . T . thermophilus HB27 cells did not require chemical treatment to induce competence, although optimal transformation was obtained by the addition of a divalent cation (Ca2+ or Mg2+) . Competence was maintained throughout the growth phase, with the highest transformation frequencies at pH 6 to 9 and at 70 degrees C . T . thermophilus HB27 and four other typical Thermus strains, T . thermophilus HB8, T . flavus AT62, T . caldophilus GK24, and T . aquaticus YT1, were also transformed to streptomycin resistance by DNA from their own spontaneous streptomycin-resistant mutants . A cryptic plasmid, pTT8, from T . thermophilus HB8 was introduced into T . thermophilus HB27 Pro- at a frequency of 10(-2).

Exp Cell Res, 1986 Apr, 163(2), 549 - 57
Polypeptides during early conjugation in Tetrahymena thermophila; Suhr-Jessen P et al.; As Tetrahymena thermophila cells differentiate from their vegetative life cycle to sexual reproduction, their polypeptide pattern undergoes a series of changes . These changes have been traced in extracellular, cellular, and subcellular compartments . The first alteration is induced by the nutritional shift-down and results in stimulation of at least one ciliary polypeptide and affects a series of polypeptides from other compartments . The second alteration is induced by mixing starved cells of complementary mating types and this stimulates the synthesis of nine ciliary polypeptides before pairs have formed and eight afterwards . At least five of these early and one of the late conjugation-related ciliary polypeptides are removed by low concentrations of EDTA, indicating that they are located on the external side of the plasma membrane . No differences were observed between polypeptides excreted during starvation and after mixing of complementary mating types . At Tris concentrations restrictive for conjugation, cilia lack the conjugation-related polypeptides . Some of these are instead found among the excreted polypeptides . Using O'Farrell gels and silver staining on isogenic cells of all possible mating types, we have been unable to correlate changes in polypeptide patterns to specific mating types.

Microbiologica, 1986 Apr, 9(2), 243 - 8
Protoplast formation and regeneration in Bifidobacterium; Brigidi P et al.; Conditions for protoplast formation and cell wall regeneration in the genus Bifidobacterium are described . The ability to form protoplast in high percentage varied according to the species considered . High reversion levels were obtained with the species B . bifidum var . pennsylvanicus, B . thermophilum and B . boum.

J Biochem (Tokyo), 1986 Apr, 99(4), 1157 - 67
Purification and properties of pyruvate kinase from Bacillus stearothermophilus; Sakai H et al.; Pyruvate kinase was purified to homogeneity from a moderate thermophile, Bacillus stearothermophilus . The molecular weight of the enzyme was found to be 250,000 on gel filtration and 242,000 on sedimentation analysis . The enzyme consisted of four identical subunits of a molecular weight of 62,000-64,000 . There were no remarkable differences between the thermophilic enzyme and mesophilic enzymes in amino acid composition, secondary structure, mono- and di-valent cation requirements for activity or specificity for nucleoside diphosphates . But the thermophilic enzyme was stable at high temperature and for a longer period of storage at lower temperature . Its specific activity was relatively high even at a low temperature (30 degrees C) . The enzyme exhibited homotropic positive cooperativity for phosphoenol-pyruvate, but not for ADP . It was allosterically activated by AMP, ribose 5-phosphate and nucleoside monophosphates, but not by fructose 1,6-bisphosphate . Activation by AMP and ribose 5-phosphate, and inhibition by inorganic phosphate were also observed even at the physiological temperature (60 degrees C) for the thermophile.

In Vitro Cell Dev Biol, 1986 Apr, 22(4), 177 - 9
Phosphate compounds as iron chelators in animal cell cultures; Rasmussen L et al.; We have studied the capacity of a number of phosphate compounds to act in the double role as a phosphate source and a detoxifier of ferric chloride hydroxo compounds, i.e . as Fe(III) chelators . The tested compounds were: orthophosphate, trimetaphosphate, alpha-glycerophosphate, beta-glycerophosphate, phytic acid, and phosphorylcholine; the test organism the ciliate protozoon Tetrahymena thermophila, an animal cell; and the nutrient medium was synthetic, consisting solely of low-molecular-weight compounds . We assessed growth rates of cells in two experimental series . First, phosphate-starved cells were exposed to the tested phosphate compound as the only phosphate source and the ferric chloride concentrations were varied stepwise from 0 to 1000 microM . Second, we offered the cells orthophosphate as a phosphate source and selected phosphate compounds as chelators . The cell growth results allow the following conclusions: orthophosphate, trimetaphosphate, alpha-glycerophosphate, and beta-glycerophosphate are excellent phosphate sources; trimetaphosphate, alpha-glycerophosphate, beta-glycerophosphate, and phytic acid are excellent Fe(III) chelators; of the tested compounds trimetaphosphate, alpha-glycerophosphate, and beta-glycerophosphate are excellent in the double role as a phosphate source and a ferric chloride hydroxo detoxifier, i.e . as a Fe(III) chelator.

Biosci Rep, 1986 Apr, 6(4), 387 - 93
DNA cytosine methylation and heat-induced deamination; Ehrlich M et al.; The heat-induced conversion of 5-methylcytosine (m5C) residues to thymine residues and of cytosine to uracil residues in single-stranded DNA was studied . The calculated rates for deamination at 37 degrees C and pH 7.4 were approximately 9.5 X 10(-10) and 2.1 X 10(-10) sec-1, respectively . N4-Methyldeoxycytidine, which is in the DNA of certain thermophilic bacteria, was more heat-resistant than was deoxycytidine and much more than was 5-methyldeoxycytidine . Thermophilic bacteria which contain N4-methylcytosine rather than m5C in their genomes may thereby largely avoid heat-induced mutation due to deamination, which is incurred by the many organisms that contain m5C in their DNA.

J Bacteriol, 1986 Apr, 166(1), 95 - 9
Temperature-sensitive binding of alpha-glucans by Bacillus stearothermophilus; Ferenci T et al.; Bacillus stearothermophilus was found to bind strongly to starch and related alpha-glucans at 25 degrees C but not at 55 degrees C . The binding at the lower temperature could be assayed either by binding of fluorescein-labeled amylopectin to washed cell suspensions or through the reversible retention of bacteria by affinity chromatography in matrices containing immobilized starch . The bacteria exhibited amylopectin-dependent agglutination . The binding and agglutination were highest in bacteria grown on substrates containing alpha-1,4-glucosidic linkages such as maltose or dextrins . The binding affinity of cells was highest for maltohexaose, lower for maltose, and low or undetectable for glucose, isomaltose, cellobiose, or lactose . The reduced binding at the higher temperature was due to the rapid breakdown of the alpha-glucosides . The bacteria exhibited an extracellular alpha-amylase activity as well as a cell-associated alpha-glucosidase with high activity at 55 degrees C but undetectable activity at 25 degrees C . The inducibility, specificity, and protease sensitivity of the thermophilic alpha-glucosidase in whole cells were similar to those of the binding activity assayed at the lower temperature . Further evidence linking the binding and alpha-glucosidase activities came from a mutant, selected through affinity chromatography, which was reduced in starch binding at room temperature and also reduced in membrane-associated alpha-glucosidase activity at 55 degrees C . These results suggest a novel survival mechanism whereby a bacterium attaches to a macromolecular substrate under nonoptimal growth conditions for possible utilization upon a shift to more favorable conditions.

Arch Biochem Biophys, 1986 Apr, 246(1), 347 - 54
Phosphatidylcholine homeostasis in phosphatidylethanolamine-depleted Tetrahymena; Smith JD; The relative contributions of the two pathways of phosphatidylcholine biosynthesis, phosphatidylethanolamine N-methyltransferase (EC 2.1.1.17) and diacylglycerol: CDP-choline cholinephosphotransferase (EC 2.7.8.1), are altered in the ciliate protozoan Tetrahymena thermophila whose phospholipid composition has been modified by culturing the organism in the presence of one of several aminophosphonic acids, as determined by measuring the incorporation of {methyl-3H}choline and {methyl-14C}methionine into phosphatidylcholine in vivo . In control cells the phosphotransferase pathway provides about 40% of the phosphatidylcholine, while in cells grown with 2-aminoethylphosphonate (AEP), 3-aminopropylphosphonate (APP), and N,N,N-trimethylaminoethyl-phosphonate (TMAEP) the contribution of the phosphotransferase pathway to phosphatidylcholine formation is 75, 90, and 26%, respectively . In AEP- and APP-grown cells, in which 80% of the phosphatidylethanolamine has been replaced by the corresponding phosphonolipid, the methyltransferase is less active since the level of the substrate phosphatidylethanolamine is reduced and neither of the phosphonolipids is a substrate for the enzyme . In TMAEP-grown cells, TMAEP competes with and reduces the incorporation of phosphocholine by the phosphotransferase pathway, leading to a smaller contribution of the pathway to phosphatidylcholine biosynthesis . The relative amounts of the two different radioactive labels incorporated into diacylphosphatidylcholine vs alkylacylphosphatidylcholine are also altered in the phosphonate-grown cells . The exogenous AEP induces a change in the glyceryl ether content of the 2-aminoethylphosphonolipid--33% in the AEP-grown cells compared to 70% in the control cells--indicating that the exogenous AEP is entering the phospholipids by the ethanolamine-phosphotransferase pathway rather than by the route of the endogenous AEP.

J Biochem (Tokyo), 1986 Apr, 99(4), 993 - 1003
Spectroscopic characterization of the ATPase of the thermophilic bacterium PS3 and its isolated subunits; Rogner M et al.; The ATPase of the thermophilic bacterium PS3, TF0F1, and its subunits has been isolated and their absorption and fluorescence spectra have been measured . The following results were obtained: The tryptophan content of the subunits was determined spectroscopically . Although tryptophan (Trp) and tyrosine (Tyr) are found in TF1, the fluorescence spectrum of native TF1 and its subunits is dominated by Tyr fluorescence; this is in contrast to other proteins . Among (native) TF1 and its subunits only TF1 and the alpha-subunit show a weak fluorescence of Trp, which is blue-shifted, indicating a location in a strongly hydrophobic environment . TF0 fluorescence is dominated by the strong Trp fluorescence . TF0F1 fluorescence is also dominated by the Trp residues . Additionally, its fluorescence is higher than the sum of the isolated TF0 and TF1, indicating marked changes in the microenvironment of the fluorescing aminoacids upon binding of TF1 to TF0.

Nucleic Acids Res, 1986 Mar 25, 14(6), 2749 - 62
In vitro deletion analysis of ARS elements spanning the replication origin in the 5' non-transcribed spacer of Tetrahymena thermophila ribosomal DNA; Amin AA et al.; Two adjacent but non-overlapping restriction fragments that encompass the replication origin of the macronuclear copy of rDNA from Tetrahymena thermophila allow autonomous replication of plasmids in the yeast Saccharomyces cerevisiae; i.e . they function as autonomously replicating segments (ARS) . Deletions generated in vitro into these fragments yield an 82 bp segment from each as the smallest sequence specifying ARS function . These 82 bp segments are at the 5' end of a 220 bp region of homology between the two original ARS restriction fragments . A 39 bp region of almost complete sequence identity between the two 82 bp fragments is suggested to be a core sequence element necessary for ARS function . This 39 bp sequence contains a region identical or nearly identical to the 11 bp yeast ARS consensus sequence (T/ATTTATPuTTTA/T) which is suggested to be essential for ARS function . Detailed comparisons of the 82 bp segments and of the 39 bp core with other ARS sequences reveal no extensive homologies aside from the consensus.

Eur J Biochem, 1986 Mar 3, 155(2), 415 - 21
Covalent linking of poly(ethyleneglycol)-bound NAD with Thermus thermophilus malate dehydrogenase . NAD(H)-regeneration unit for a coupled second-enzyme reaction; Eguchi T et al.; Poly(ethyleneglycol)-bound NAD (PEG-NAD) was covalently linked to Thermus thermophilus malate dehydrogenase with a bifunctional reagent, 3,3'-(1,6-dioxo-1,6-hexanediyl)bis-2-thiazolidinethione . The covalently linked malate-dehydrogenase--PEG--NAD complex (MDH-PEG-NAD) was purified by DEAE-Sephadex column chromatography to remove unbound PEG-NAD, and fractionated by blue-Sepharose column chromatography into four preparations: MDH-PEG-NAD I, MDH-PEG-NAD II, MDH-PEG-NAD III and MDH-PEG-NAD IV . The average numbers of NAD moieties covalently bound per subunit of MDH-PEG-NAD I, MDH-PEG-NAD II, MDH-PEG-NAD III and MDH-PEG-NAD IV were 1.2, 1.2, 0.8 and 0.5, respectively, and the values were confirmed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis . 60-80% bound NAD moiety of these preparations of MDH-PEG-NAD was reduced by the enzyme moiety in the presence of L-malate, and the specific activity of the enzyme moiety of the preparations was more than 80% that of the native enzyme . MDH-PEG-NAD I has the following properties . The Km value for exogenous NAD is three times that of the native enzyme . The coenzyme activity of its NAD moiety is 20-40% that of native NAD for alcohol and lactate dehydrogenases . The complex catalyzes the oxidation of L-malate in the presence of the redox system of 5-ethylphenazinium ethyl sulfate and a tetrazolium salt with a rate constant of 0.11 s-1 . The coenzyme moiety of the complex can also be recycled by coupled reactions of the active site of the same complex and alcohol dehydrogenase . These results indicate that MDH-PEG-NAD works as an NAD(H)-regeneration unit for coupled reactions.

Dev Biol, 1986 Mar, 114(1), 72 - 86
Intracellular pattern reversal in Tetrahymena thermophila . II . Transient expression of a janus phenocopy in balanced doublets; Frankel J et al.; Homopolar doublets of Tetrahymena thermophila which have two normal oral systems directly opposite one another may undergo a global transformation of cell surface geometry to create transient imitations of mirror-image configurations brought about by mutations at janus gene loci . The process by which a typical doublet transforms into a janus-like organization involves loss of capacity to form oral structures at one of the two normal oral meridians, followed by interpolation of reversed oral structures at a new location to the cell's right of the disappearing normal oral meridian . At the same time, the contractile vacuole pore (CVP) set on the side of the cell that is undergoing the transformation shifts to the left . The combination of these events creates a symmetrical large-scale organization in which both of the CVP sets are situated on one side of the cell, between the normal and the partially reversed oral apparatus . This unilateral positioning of CVP sets is commonly manifested even when reversed oral structures are absent . These configurations probably represent intermediate stages in the transformation of balanced typical doublets into singlets . We propose that this pathway of regulation from the doublet to the singlet state, like the more common one that starts from unbalanced typical doublets (described in the preceding paper), involves reverse intercalation . The remarkable resemblance between the transient configuration described here and the stable configuration of janus mutant cells leads us to suggest that the phenotype of the mutant is also a consequence of reverse-intercalation, in that case provoked by a loss of capacity to maintain positional values rather than by a geometrical instability in the system of positional values.

Dev Biol, 1986 Mar, 114(1), 53 - 71
Intracellular pattern reversal in Tetrahymena thermophila . I . Evidence for reverse intercalation in unbalanced doublets; Nelsen EM et al.; Homopolar doublets of Tetrahymena thermophila possess two sets of similar cell surface structures, the most prominent of which are the complex and asymmetrical oral apparatuses . These initially are located on opposite surfaces of the duplex cell, but tend to shift so that they are no longer directly opposite each other . The two sets of oral structures are then separated by one wider and one narrower arc of cell surface . When one arc becomes sufficiently narrow, a new third oral apparatus with partially reversed internal asymmetry frequently becomes interposed between the two preexisting oral apparatuses, always within the narrower arc . After this happens, the reliability of development of new oral structures, particularly of the interposed ones, is reduced . Contractile vacuole pores, typically present within both arcs of homopolar doublets, tend to disappear from the narrower arcs . This anomalous partial triplet condition appears to be a transient intermediate stage in the reversion of homopolar doublets to normal singlets . We interpret the interposition of a transient third oral system in doublets that are regulating toward the singlet state as being a consequence of reverse intercalation of new positional values subsequent to excessive crowding of the preexisting positional values . This interpretation is an adaptation of the shortest-distance intercalation rule of the polar coordinate model applied in an intracellular and morphallactic context.

Mol Cell Biol, 1986 Mar, 6(3), 900 - 5
Macronuclear DNA molecules of Tetrahymena thermophila; Conover RK et al.; The physical organization of the DNA in the macronuclei of Tetrahymena thermophila was investigated by using alternating-orthogonal-field gel electrophoresis . The genome consisted of a spectrum of molecules with lengths ranging from less than 100 to in excess of 1,500 kilobase pairs . There were about 270 different macronuclear DNA molecules, with an average size of about 800 kilobase pairs . Specific genes were mapped and were generally found on macronuclear DNA molecules of the same size in different strains of T . thermophila . This indicates that the molecular mechanisms giving rise to the macronuclear DNA molecules were precise . The fragmentation process that gave rise to macronuclear DNA molecules occurred between 11 and 19 h after the initiation of conjugation.

Hokkaido Igaku Zasshi, 1986 Mar, 61(2), 314 - 6
{Farmer's lung in the east of Hokkaido . A follow-up study in 1985}; Konno T et al.; The follow-up study of 16 patients with farmer's lung and 35 farmers having antibody to thermophilic actinomyces was performed . The following results were obtained . Three patients out of 16 patients with farmer's lung were readmitted to the hospital . The patients out of 11 believed that the dust respirator is useful for prevention against the development of farmer's lung . Any new patient was not found out from farmers having antibody to thermophilic actinomyces in 1985.

Exp Cell Res, 1986 Mar, 163(1), 165 - 74
Proteolytic response to nutritional step-down in Tetrahymena; Jonassen TO et al.; The ciliate Tetrahymena thermophila is usually grown in a medium containing proteose peptone and yeast extract as organic nutrients . When the ciliate is transferred to step-down conditions, i.e., an inorganic medium, it is shown that the cells respond by rapidly and drastically increasing their rate of protein degradation . A method for measuring the response to step-down conditions is presented, and the response is characterized . The types of proteinases involved are indicated by the use of specific inhibitors . It is concluded that Tetrahymena reacts in much the same way as mammalian cells, and provides a suitable system for investigating the regulation of protein degradation.

Arch Biochem Biophys, 1986 Feb 15, 245(1), 8 - 13
The use of dithionite reduction to identify the essential tyrosine residue in the F1-ATPase from the thermophilic bacterium, PS3, that reacts with 7-chloro-4-nitrobenzofurazan; Verburg JG et al.; When the F1-ATPase from the thermophilic bacterium, PS3, was inactivated by greater than 90% with 7-chloro-4-nitro{14C}benzofurazan ({14C}Nbf-Cl) at pH 7.4, 1.4 mol of {14C}Nbf were incorporated per mol of enzyme . After pepsin digestion of the labeled enzyme at pH 3.0, a single, major peak of radioactivity was resolved by reversed-phase high-performance liquid chromatography under acidic conditions were peptidyl Nbf-O-tyrosine is stable . This radioactive peak, designated RP-1, eluted with a retention time of 95 min . When the material in RP-1 was subjected to reversed-phase high-performance liquid chromatography under the same conditions after treatment with sodium dithionite, a single, major peak of radioactivity, designated RP-2, was resolved with a retention time of 52 min . Automatic Edman degradation of this material revealed that it has the amino acid sequence I-Y*-V-P-A-D-(D), where Y* presumably represents peptidyl {14C}Nbf-O-tyrosine . These results provide the basis for a facile method to purify peptides containing {14C}Nbf-O-tyrosine in which the labeled residues can be identified by amino acid sequence analysis using the Edman degradation.

Nucleic Acids Res, 1986 Feb 11, 14(3), 1365 - 78
Secondary structure of Tetrahymena thermophilia 5S ribosomal RNA as revealed by enzymatic digestion and microdensitometric analysis; Sneath B et al.; The secondary structure of {32P} end-labeled 5S rRNA from Tetrahymena thermophilia (strain B) has been investigated using the enzymes S1 nuclease, cobra venom ribonuclease and T2 ribonuclease . The results, analyzed by scanning microdensitometry and illustrated by three-dimensional computer graphics, support the secondary structure model of Curtiss and Vournakis for 5S rRNA . Aberrent mobility of certain RNA fragments on sequencing gels was observed as regions of band compression . These regions are postulated to be caused by stable internal base-pairing . The molecule was probed with T2 RNase in neutral (pH 7.5) and acidic (pH 4.5) buffers and only minor structural differences were revealed . One of the helices was found to be susceptible to enzymatic attack by both the single-strand and double-strand specific enzymes . These observations are evidence for the existence of dynamic structural equilibria in 5S rRNA.

Am J Vet Res, 1986 Feb, 47(2), 254 - 8
Epidemiologic study of campylobacteriosis in Iowa cattle and the possible role of unpasteurized milk as a vehicle of infection; Warner DP et al.; Bile samples were collected from 477 Iowa dairy cows and were cultured for thermophilic campylobacters . The prevalence of thermophilic campylobacters in the bile was 15.5% . Campylobacter jejuni and C coli from dairy cattle, chickens, pigs, sheep, and human beings were serotyped to develop host-species profiles . Human and cattle serologic profiles were the most similar, and human and chicken profiles shared several similarities . Epidemiologic data from 168 human cases of campylobacteriosis indicated that 23% of the cases were associated with consumption of unpasteurized milk.

Exp Cell Res, 1986 Feb, 162(2), 390 - 400
Analysis of nuclei from exponentially growing and conjugated Tetrahymena thermophila using the flow microfluorimeter; Brunk CF et al.; Isolated nuclei of Tetrahymena thermophila from both exponentially growing cultures and from cells following conjugation have been analysed using a flow microfluorimeter . The macronuclei from a culture in exponential growth display a single broad distribution of DNA contents, without bimodal character . The micronuclei are virtually all in G2 phase (4C) . The mean of the macronuclear DNA distribution is about 12.4 times the micronuclear mean (50C) . When cells are starved in preparation for conjugation, the macronuclei DNA content is decreased about 30%, but the distribution remains similar to that of nuclei from a culture in exponential growth . Following conjugation, the macronuclear anlagen develop through a set of relatively synchronous endoreplications . At 12 h after the initiation of conjugation the anlagen are at a 4C stage and at 18 h they are virtually all at a 8C stage . If the culture is refed, anlagen development progresses to a 16C and 32C, but the synchrony is poorly conserved . Cells that are not refed are arrested at the 8C stage and only a fraction of the population ever become mature macronuclei . In general we do not observe distinct peaks of anlagen with DNA contents in excess of 32C . The amitotic division of macronuclei may obscure any endoreplications producing anlagen stages with higher DNA content.

J Biochem (Tokyo), 1986 Feb, 99(2), 349 - 56
Methylation and demethylation of solubilized chemoreceptors from thermophilic bacterium PS-3; Hirota N; Methyl-accepting chemotaxis proteins (MCPs) were solubilized from the membrane of thermophilic bacterium PS-3 in the presence of Triton X-100 . The solubilized MCPs could be methylated and demethylated . Methylation of the solubilized MCPs reached a steady state, at which the methylation and demethylation rates were equal . The solubilized MCPs were purified by anti-MCPs Sepharose 4B column chromatography . The purified MCPs could also be methylated and demethylated without reconstituting them into liposomes . As suggested by the results of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis of the purified MCPs, ion-exchange chromatography showed that MCPs consisted of at least two components . Each component appeared on SDS gel electrophoresis as multiple bands in the 64K to 70K dalton range or in the 70K to 84K dalton range . The initial rate and level of methylation of the solubilized MCPs were increased by the addition of attractants: glutamate, L-serine, L-aspartate, D-glucose, etc . The threshold of the glutamate concentration for this increase was about 10(-7) M . The rate of demethylation was also increased by attractants.

J Cell Sci, 1986 Feb, 80, 57 - 73
Freeze-fracture analysis of thylakoid membranes and photosystem I and II enriched fractions from Phormidium laminosum; Glick RE et al.; Thylakoid membranes of the thermophilic cyanobacterium Phormidium laminosum have been fractionated into photosystem II and photosystem I particles . These fractions have been characterized by their partial electron transport activities, and biochemical and spectral properties . Exoplasmic fracture face and protoplasmic fracture face particles in the unfractionated thylakoid membranes were shown to correspond in size to particles in freeze-fractured photosystem II and photosystem I fractions, respectively . Differences between the histograms of the thylakoid membrane protoplasmic fracture face particles and the isolated photosystem I particles suggest that in addition to photosystem I complexes some of the particles on the thylakoid protoplasmic fracture face may be related to cytochrome b/f complexes, the hydrophobic component of the coupling factor, or respiratory complexes.

J Protozool, 1986 Feb, 33(1), 30 - 8
Alternative processing of sequences during macronuclear development in Tetrahymena thermophila; White TC et al.; DNA is eliminated during development of the somatic MACronucleus from the germinal MICronucleus in the ciliated protozoan, Tetrahymena thermophila . Facultatively persistent sequences are a class of sequences that persist in the MAC DNA of some cell lines but are eliminated from the MAC DNA of other cell lines . One cloned MAC fragment contains a persistent sequence as well as sequences normally retained in the MAC . When this cloned fragment was used to construct MAC restriction maps of this region in cell lines whose MAC DNAs do, or do not, contain the persistent sequence, extensive variation in the map flanking this region was observed . The different DNA rearrangements of this MIC segment are epigenetically determined during or soon after MAC development . Moreover, different rearrangements may occur among the 45 copies of this MIC segment as a MAC is formed, resulting in polymorphisms that are later resolved by phenotypic assortment.

Proc Natl Acad Sci U S A, 1986 Feb, 83(3), 576 - 80
Isolation of a thermostable enzyme variant by cloning and selection in a thermophile; Liao H et al.; We developed a method for rapidly generating thermostable enzyme variants . Our strategy is to introduce the gene coding for a given enzyme from a mesophilic organism into a thermophile, Bacillus stearothermophilus . Variants that retain the enzymatic activity at the higher growth temperatures of the thermophile are then selected . This strategy was applied to kanamycin nucleotidyltransferase, which confers resistance to the antibiotic kanamycin . B . stearothermophilus carrying the wild-type enzyme is resistant to the antibiotic at 47 degrees C but not at 55 degrees C and above . Variants that were kanamycin resistant at 63 degrees C were obtained by selection of spontaneous mutants, by passage of a shuttle plasmid through the Escherichia coli mutD5 mutator strain and introduction into B . stearothermophilus by transformation, and by growing the thermophile in a chemostat . The kanamycin nucleotidyltransferases purified from these variants were all more resistant to irreversible thermal inactivation than is the wild-type enzyme, and all have the same single amino acid replacement, aspartate to tyrosine at position 80 . Mutants that are even more heat stable were derived from the first variant by selecting for kanamycin resistance at 70 degrees C, and these carry the additional change of threonine to lysine at position 130 . This strategy is applicable to other enzymatic activities that are selectable in thermophiles or that can be screened for by plate assays.

Arch Microbiol, 1986 Feb, 144(1), 102 - 4
N2 fixation and NH4+ assimilation in the thermophilic anaerobes Clostridium thermosaccharolyticum and Clostridium thermoautotrophicum; Bogdahn M et al.; Inorganic nitrogen metabolism in the obligate anaerobic thermophiles Chlostridium thermosaccharolyticum and Clostridium thermoautotrophicum differs in several respects . C . thermosaccharolyticum contains a nitrogenase as inferred from NH4+ repressible C2H2 reduction, a glutamine synthetase which is partially repressed by ammonium, very labile glutamate synthase activities with both NADH and NADPH, NADPH-dependent glutamate dehydrogenase, and NH4+-dependent asparagine synthetase . C . thermoautotrophicum contains no nitrogenase, but glutamine synthetase, no glutamate synthase, no glutamate dehydrogenase, but a NADH-dependent alanine dehydrogenase and a NH4+-dependent asparagine synthetase.

Arch Biochem Biophys, 1986 Feb 1, 244(2), 865 - 71
Sequence of the radioactive tryptic peptide obtained after inactivating the F1-ATPase of the thermophilic bacterium PS3 with 5'-p-fluorosulfonylbenzoyl{3H}adenosine at 65 degrees C; Bullough DA et al.; Following a lag of about 30 min, the F1-ATPase from the thermophilic bacterium, PS3 (TF1), was inactivated slowly by 0.8 mM 5'-p-fluorosulfonylbenzoyladenosine (FSBA) at 23 degrees C and pH 7.0 . When the enzyme was treated with 0.2 mM FSBA at pH 7.0 and 23 degrees C for 15 min and gel-filtered, no enzyme activity was lost . However, the lag in inactivation was abolished when the enzyme was subsequently incubated with 2.0 mM FSBA at 23 degrees C in the pH range from 6.8 to 10.0 . The pH-inactivation profile obtained under these conditions revealed a pK alpha of about 9.3 which was associated with the inactivation . When pretreated TF1 was inactivated at 23 degrees C with {3H}FSBA by about 90%, greater than 20 mol of {3H}SBA was incorporated per mole of enzyme . TF1 was inactivated rapidly by 0.8 mM FSBA at pH 6.4 and 65 degrees C, and no lag was observed . Following inactivation of TF1 with 0.8 mM {3H}FSBA at 65 degrees C and pH 6.4, about 10 mol of {3H}SBA was incorporated per mole of enzyme . When a tryptic digest of the labeled enzyme was fractionated by reversed-phase high-performance liquid chromatography, a single major radioactive peptide was isolated . When subjected to automatic Edman degradation, this peptide was shown to have the amino acid sequence: A-L-A-P-E-I-V-G-E-E-H-X-Q-V-A-R, where X indicates that a phenylthiohydantoin derivative was not detected in cycle 12 . However, from the DNA sequence of the gene encoding the subunit of TF1 (Y . Kagawa, M . Ishizuka, T . Saishu, and S . Nakao (1985) Abstracts International Symposium on Energy Transducing ATPases, Kobe, Japan, p . 84), this position has been shown to be occupied by tyrosine . This tyrosine is homologous with beta-Tyr-368 of the bovine mitochondrial F1-ATPase (MF1) the modification of which is responsible for the inactivation MF1 by FSBA.

J Bacteriol, 1986 Feb, 165(2), 595 - 601
Phosphorylation of proteins in Clostridium thermohydrosulfuricum; Londesborough J; Cell extracts of the thermophile Clostridium thermohydrosulfuricum catalyzed the phosphorylation by {gamma-32P}ATP of several endogenous proteins with Mrs between 13,000 and 100,000 . Serine and tyrosine were the main acceptors . Distinct substrate proteins were found in the soluble (e.g., proteins p66, p63, and p53 of Mrs 66,000, 63,000, and 53,000, respectively) and particulate (p76 and p30) fractions, both of which contained protein kinase and phosphatase activity . The soluble fraction suppressed the phosphorylation of particulate proteins and contained a protein kinase inhibitor . Phosphorylation of p53 was promoted by 10 microM fructose 1,6-bisphosphate or glucose 1,6-bisphosphate and suppressed by hexose monophosphates, whereas p30 and p13 were suppressed by 5 microM brain (but not spinach) calmodulin . Polyamines, including the "odd" polyamines characteristic of thermophiles, modulated the labeling of most of the phosphoproteins . Apart from p66, all the proteins labeled in vitro were also rapidly labeled in intact cells by 32Pi . Several proteins strongly labeled in vivo were labeled slowly or not at all in vitro.

Science, 1986 Jan 31, 231(4737), 470 - 5
The intervening sequence RNA of Tetrahymena is an enzyme; Zaug AJ et al.; A shortened form of the self-splicing ribosomal RNA (rRNA) intervening sequence of Tetrahymena thermophila acts as an enzyme in vitro . The enzyme catalyzes the cleavage and rejoining of oligonucleotide substrates in a sequence-dependent manner with Km = 42 microM and kcat = 2 min-1 . The reaction mechanism resembles that of rRNA precursor self-splicing . With pentacytidylic acid as the substrate, successive cleavage and rejoining reactions lead to the synthesis of polycytidylic acid . Thus, the RNA molecule can act as an RNA polymerase, differing from the protein enzyme in that it uses an internal rather than an external template . At pH 9, the same RNA enzyme has activity as a sequence-specific ribonuclease.

J Biol Chem, 1986 Jan 25, 261(3), 1071 - 6
Nonrandom utilization of acetylation sites in histones isolated from Tetrahymena . Evidence for functionally distinct H4 acetylation sites; Chicoine LG et al.; Macro- and micronuclei of the ciliated protozoan Tetrahymena thermophila afford a unique opportunity to study histone acetylation under conditions where postsynthetic "transcription"-related acetylation and synthetic "deposition"-related acetylation are nonoverlapping . Recent studies have demonstrated that at least two general systems of acetylation operate in Tetrahymena . One is postsynthetic, macronuclear specific, and may be related to gene expression in that nucleus (Vavra, K . J., Allis, C . D., and Gorovsky, M . A . (1982) J . Biol . Chem . 257, 2591-2598) . The other is synthetic, common to macro- and micronuclei, and is likely related to histone deposition during replication (Allis, C . D., Chicoine, L . G., Richman, R., and Schulman, I . G . (1985a) Proc . Natl . Acad . Sci . U . S . A., 82, 8048-8052) . A unique feature of H3 and H4 in Tetrahymena is that neither are blocked at their amino termini . We have exploited this fact as well as the resolving capability of acid-urea gel electrophoresis and current microsequencing techniques to examine whether utilization of different NH2-terminal acetylation sites is random or nonrandom during the progression toward high acetylation states . Of the four acetylation sites which have been identified in H4 (in Tetrahymena these are lysines at positions 4, 7, 11, and 15), we find that lysine 7 is the exclusive site of postsynthetic acetylation in populations of monoacetylated H4 isolated from macronuclei . This site is retained in populations of diacetylated H4, which are acetylated exclusively at lysines 4 and 7 . Our data also suggest that there is some preference for using lysine 11 (as compared to 15) as the third site of acetylation in triacetylated molecules . The data demonstrate that the postsynthetic acetylation-deacetylation process is surprisingly nonrandom for H4 in Tetrahymena macronuclei . We have also investigated the same question with macronuclear H3 (which contains acetylation sites at lysines 9, 14, 18, and 23) . Our data demonstrate that unlike H4, lysines at position 9 or 14 are likely to be utilized as sites of acetylation within a population of monoacetylated H3 . Both of these acetylation sites are retained in diacetylated H3 which suggests that if site 9 is used initially as the site of monoacetylation, 14 is used secondarily (and vice versa) . Our data show, moreover, that there is a preference for utilizing lysine 18 as the third acetylation site (in triacetylated H3) . Thus, these data show that H3 is also acetylated in a nonrandom fashion in macronuclei . Finally, we have determined which acetylation sites are utilized in macro- or micronuclear H4 when it is undergoing active synthesis and deposition.(ABSTRACT TRUNCATED AT 400 WORDS)

J Biol Chem, 1986 Jan 5, 261(1), 363 - 9
Chromatin structure of the telomeric region and 3'-nontranscribed spacer of Tetrahymena ribosomal RNA genes; Budarf ML et al.; The chromatin structure of the 3'-nontranscribed spacer of the linear rRNA gene molecules of Tetrahymena thermophila was examined . This region includes the transcription termination site, two sets of recently identified conserved spacer repeats (Type IV and V repeats (Challoner, P . B., Amin, A . A., Pearlman, R . E., and Blackburn, E . H . (1985) Nucleic Acids Res . 13, 2661-2680}, and the terminus of the molecule . Using sensitivity to nucleases as a probe, a unique chromatin structure was found in this rDNA region . Proceeding from the end of the rDNA molecule, the telomeric repeated sequence, (CCCCAA)n, was packaged in a non-nucleosomal complex adjacent to three phased nucleosomes . This nucleosomal structure was disrupted at the Type V repeat region, which, compared with the neighboring nucleosomal region, was more accessible to nucleases and, from both micrococcal nuclease and DNase I digestion results, was packaged in chromatin differently from the sequences flanking it on both sides . The region between the Type V repeats and adjacent to the transcription termination site was in yet another distinguishable chromatin structure as judged by its sensitivity to nucleases . It includes sites protected in chromatin and sites which were cleaved in chromatin but not detectably digested in DNA controls, suggesting that specific proteins are also associated with this region.

Basic Res Cardiol, 1986, 81 Suppl 1, 95 - 102
Myothermal economy of rat myocardium, chronic adaptation versus acute inotropism; Holubarsch C et al.; By means of rapid planar Hill type antimony-bismuth thermophiles the initial heat liberated by papillary muscles was measured synchronously with developed tension for control (C), pressure-overload (GOP), and hypothyrotic (PTU) rat myocardium (chronic experiments) and after application of 10(-6) M isoproterenol or 200 10(-6) M UDCG-115 . Economy of force production was analyzed by the ratio of initial heat versus developed tension-time integral . This ratio was found to be reduced by 34% in GOP and by 43% in PTU myocardium (P less than 0.01, respectively) indicating increased economy of force production . In contrast, isoproterenol increased initial heat versus tension-time integral by 70% (P less than 0.01) indicating reduced economy of force production . No change in this ratio was found for UDCG-115 . The presented data indicates that long and short term modulation of myocardial energetic costs of force generation is possible . The basic mechanisms for these myocardial alterations are discussed.

Gene, 1986, 44(1), 139 - 42
Molecular cloning of the gene encoding the valyl-tRNA synthetase from Bacillus stearothermophilus; Brand NJ et al.; The valS gene from the thermophile Bacillus stearothermophilus encoding the valyl-tRNA synthetase has been cloned on a 13.8-kb plasmid . The gene product and its kinetic properties are comparable with those of the native enzyme.

J Basic Microbiol, 1986, 26(7), 371 - 82
Effect of methyl viologen and oxygen concentration on thermophilic bacteria; Allgood GS et al.; The level of superoxide dismutase, catalase, and peroxidase was determined in obligately thermophilic bacteria after growth either in the presence of methyl viologen (paraquat) or exposed to an increased atmospheric oxygen concentration . In general, the superoxide dismutase level in these organisms was not altered by paraquat addition and had a varied response to oxygenation . Incorporation of paraquat in the growth medium resulted in minimally three-fold higher levels of catalase . The peroxidase level varied as a response to oxygen stress whereas cellular catalase levels were generally higher.

Dev Genet, 1986, 6(3), 213 - 38
How the mirror-image pattern specified by a janus mutation of Tetrahymena thermophila comes to expression; Frankel J et al.; The initial changes of cell-surface organization that occurred as the recessive janA1 (janus) mutation of Tetrahymena thermophila first became expressed were elucidated in a special mating scheme in which old macronuclei homozygous for janA+ were synchronously replaced by new macronuclei homozygous for janA1 . During this period of onset of expression, the number, regularity, and asymmetry of the ciliary rows remained unchanged . New normal (primary) oral apparatuses (OAs) continued to be formed posterior to old OAs, as in normal cells . At about four fissions after conjugation, abnormal (secondary) OAs with a partial reversal of asymmetry began to appear nearly opposite to the primary OAs, close to but not at the eventual circumferential position of janA1 secondary OAs . The array of contractile vacuole pores (CVPs), normally located adjacent to two ciliary rows centered near 22% of the cell circumference to the right of the primary oral meridian, underwent a two-step transformation: first, the number of adjacent ciliary rows bearing CVPs increased to 3, 4, and sometimes 5, then "skipped" rows appeared within this broadened CVP-arc to split the single set of CVPs into two separated subsets . The CVP transformations occurred gradually and progressively . They began prior to the expression of secondary OAs but accelerated as secondary OAs appeared . As the CVP arc became broader, its midpoint shifted somewhat to the right, away from the primary oral meridian, but ended up close to halfway between the primary and secondary oral meridians . The data provide a better fit to an intercalation model than to an alternative double-gradient model, suggesting that the janA1 mutation alters the large-scale organization of positional values by preventing the expression of a subset of these values and thus provoking reverse-intercalation of the remainder.

Curr Genet, 1986, 10(7), 537 - 44
Immunological homologies between ribosomal proteins amongst lower eukaryotes; Kreutzfeldt C et al.; Polyclonal antibodies were raised against the purified ribosomal proteins L1 and L2, the 5S rRNA binding protein L3, all from Saccharomyces cerevisiae, and against L1 and L2 from Schizosaccharomyces pombe (numbering according to Otaka and Osawa 1981; Otaka et al . 1983, respectively) . For clarity prefixes Sc and Sp have been added to the numbering of proteins derived from S . cerevisiae and S . pombe, respectively . Ribosomal proteins from these yeasts and from Kluyveromyces marxianus, Rhodotorula glutinis, the slime mold Dictyostelium discoideum and the protozoan Tetrahymena thermophila were checked for antigenic cross-reactivity by the immunoblot technique . Anti-ScL1 bound to the largest ribosomal proteins of all organisms but not with equal strength . A fast migrating protein band from R . glutinis was also reactive . Anti-ScL2 reacted strongly with L2 or analogous proteins derived exclusively from the yeasts . Anti-ScL3 cross-reacted only with one protein band from K . marxianus, whereas anti-SpL1 cross-reacted with L1 or its analogues from the other organisms, but also with proteins of lower molecular weight . In S . cerevisiae, these proteins are located exclusively on the small ribosomal subunit . L2 or analogous ribosomal proteins of all organisms were recognized by anti-SpL2 but additionally the ribosomal protein YL28 of S . cerevisiae and fast migrating proteins of T . thermophila exhibited anti-SpL2 binding.

J Cell Biochem, 1986, 31(3), 195 - 202
Identification of a biochemical marker for the secretory pathway in Tetrahymena thermophila; Maihle NJ et al.; Tetrahymena thermophila is a ciliated protozoan studied by investigators from a wide range of disciplines as a model system for a variety of specialized eukaryotic cell functions . The proteinaceous secretory products of T thermophila have been isolated and characterized and in this study we identify the major secretory product, a 34,000 Mr polypeptide, and use an antiserum prepared against this secretory protein to (1) demonstrate that this 34,000 Mr polypeptide is truly a secreted protein in Tetrahymena and (2) monitor the synthesis and transport of this protein by indirect immunofluorescence and light microscopy during mucocyst biogenesis.

Nucleic Acids Symp Ser, 1986, (17), 175 - 8
Effects of modification of 4-thiouridine in E . coli tRNAfMet on methylation reaction with a thermostable Gm-methylase; Hori H et al.; Gm-methylase isolated from an extreme thermophile, Thermus thermophilus HB 27 methylates the 2'-OH of the ribose of G18 in the consensus GG sequence in the D loop, by recognizing the D loop-and-stem structure as a minimal substrate . Modification of s4U8 of E . coli tRNAfMet with S-benzylthioisothiourea resulted in a considerable decrease in methylation activity of Gm-methylase . The effect was cancelled by reduction with beta-mercaptoethanol . However, aminoacylation activity and methylation activity of m1A-methylase were scarcely influenced by the modification . These results suggest the involvement of s4U8 residue of tRNA in the recognition of Gm-methylase.

Arch Biochem Biophys, 1986 Jan, 244(1), 254 - 60
Isolation of an intrinsic antenna chlorophyll a-protein from the photosystem I reaction center complex of the thermophilic cyanobacterium Synechococcus sp; Sonoike K et al.; A chlorophyll-protein was isolated from a Synechococcus P700-chlorophyll a-protein complex free from small subunits (CP1-e) by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis after treatment with 2% 2-mercaptoethanol and 2% SDS . In contrast to CP1-e which, when electrophoresed under denaturating conditions, showed two polypeptide bands of 62 and 60 kDa, the chlorophyll-protein contained only the 60-kDa polypeptide and hence is called CP60 . The yield of CP60 was maximal with 1-2% SDS and 2-4% sulfhydryl reagents because the chlorophyll-protein was denatured at higher concentrations of the reagents . The absorption spectrum of CP60, which retained more than half of the chlorophyll alpha molecules originally associated with the 60-kDa subunit of the photosystem I reaction center complex, showed a red band maximum at 672 nm and a small absorption band around 700 nm at liquid nitrogen temperature . CP60 emitted a fluorescence band at 717 to 725 nm at 77 degrees K . The temperature dependence of the far red band of CP60 was essentially the same as that of CP1-e between 77 and 273 degrees K . No photoresponse of P700 was detected in CP60 . The results suggest that the two polypeptides resolved by SDS-gel electrophoresis from CP1-e are apoproteins of two distinct chlorophyll-proteins and that CP60 represents a chlorophyll-bearing 60-kDa subunit functioning as an intrinsic antenna protein of the photosystem I reaction center complex . It will also be shown that the temperature dependence of the far red fluorescence band is not related to the photosystem I photochemistry.

J Mol Evol, 1986, 24(1-2), 143 - 51
Characterization of the macronuclear DNA of different species of Tetrahymena; Conover RK et al.; The macronuclear DNAs from 20 different species of Tetrahymena were characterized using Alternating Orthogonal Field (AOF) gel electrophoresis . Each species has approximately 300 different macronuclear DNA molecules that range in size from about 100-2000 kb pairs . Although the individual macronuclear DNA molecules are not well resolved on an AOF gel, most species have a unique profile of macronuclear DNA . The sequences that hybridize with histone H4 (Tetrahymena) and ubiquitin (yeast) genes were identified on the separated macronuclear DNA molecules of the different species . All species have 2 histone H4 genes located on macronuclear DNA molecules of different sizes . This is consistent with the duplication of the histone H4 gene prior to the speciation events leading to the various species of Tetrahymena . The number and sizes of the macronuclear DNA molecules that hybridize with the ubiquitin probe vary from species to species . A grouping of the different species of Tetrahymena based on this hybridization pattern parallels groupings of the species based on ribosomal RNA sequences and isoenzymes . Some intraspecific variation among different strains of Tetrahymena thermophila was detected using ubiquitin and 5S ribosomal RNA as probes.

Mol Gen Genet, 1986 Jan, 202(1), 169 - 71
Complete nucleotide sequences of Bacillus plasmids pUB110dB, pRBH1 and its copy mutants; Muller RE et al.; The deletion plasmids, pRBH1 (1.5 MDa, kanamycin resistance, Kmr) and pUB110dB (1.5 MDa, Kmr), were obtained from pTB913 (2.9 MDa, Kmr, isolated from a thermophilic bacillus) and pUB110 (3.0 MDa, Kmr, from Staphylococcus aureus), respectively . All the nucleotide sequences of these deletion plasmids were determined . Replication origin regions of pRBH1 and pUB110dB contained, respectively, 63 base-pair inverted repeat and a large open reading frame, encoding RepB protein (235 amino acid residues) . The nucleotide sequences were identical to each other except for one base in the center of the inverted repeat . Two copy number mutant plasmids, pRBHC3 and pRBHC7, were obtained from pRBH1 . The mutation points were located at different positions in the RepB protein coding region (Gly to Asp for pRBHC3 and Gly to Glu for pRBHC7) . RepB protein was shown to be involved in the copy number control of these plasmids.

Nucleic Acids Symp Ser, 1986, (17), 199 - 201
Circular dichroism analyses of tRNA X protein interactions; Yokoyama S et al.; The interactions of tRNA species with aminoacyl-tRNA synthetases and polypeptide chain elongation factor Tu from Thermus thermophilus HB8 were studied by the analyses mainly of the circular dichroism band of 2-thioribothymidine in position 54 of T . thermophilus tRNA species.

J Biol Chem, 1985 Dec 25, 260(30), 16424 - 32
The structure of manganese superoxide dismutase from Thermus thermophilus HB8 at 2.4-A resolution; Stallings WC et al.; An atomic model of tetrameric manganese superoxide dismutase from Thermus thermophilus HB8 has been built into an electron density map at 2.4 A resolution, using chemical sequences of Mn dismutases from Thermus aquaticus and Bacillus stearothermophilus . The monomer fold is structurally very similar to the fold of iron dismutase and comprises two domains, each contributing two ligands to the metal . The Mn(III) ion is bound by protein ligands assigned as His 28, His 83, Asp 165, and His 169 . Near neighbors in the metal-ligand environment include a series of hydrophobic residues, Phe 86, Trp 87, Trp 131, and Trp 167 . The hydroxyl groups of two Tyr residues, at 36 and 182, are less than 7 A from the metal, as is His 32 . Gln 150 forms a bridge between Tyr 36 and Trp 131 . These ligands and nearby residues are strongly conserved in the known sequences of Mn dismutases . Only one of the two oxygens of Asp 165 has been assigned as a metal ligand, so that in the current model four protein atoms bind Mn(III) . These ligand atoms form part of an approximate trigonal bipyramid in which water may occupy an axial position on the side opposite His 28 . The conformation of the protein is unusual in the vicinity of the first ligand, His 28, as a consequence of the insertion of an extra residue in an alpha-helix . The distortion of the helix allows His 32 to stack against the ligand, His 169, and brings Tyr 36 close to the Mn ion . Across one of the dimer interfaces, the two Mn ions are separated by about 18 A, and active center residues from adjoining subunits interdigitate; Tyr 172 interacts with His 32 of the neighboring chain and Glu 168 with the backbone of 168 and with the ligand His 169 from the opposite subunit . Only one other dimer interface occurs in the tetramer; it involves residues 55-62 and sequences near 140 and 156 . The center of the oligomeric molecule is filled with solvent.

J Biol Chem, 1985 Dec 5, 260(28), 15298 - 303
Screening for thermostable mutant of kanamycin nucleotidyltransferase by the use of a transformation system for a thermophile, Bacillus stearothermophilus; Matsumura M et al.; A structural gene of kanamycin nucleotidyltransferase cloned into a single-stranded bacteriophage M13 was subjected to mutagenesis with hydroxylamine . Having recloned the mutagenized gene of the enzyme in a vector plasmid pTB922, the recombinant plasmid was used to transform Bacillus stearothermophilus with a purpose of screening for the more thermostable enzyme than the wild type . Out of greater than 8 X 10(3) transformants, 12 clones that were suspected to harbor the mutant gene encoding the more thermostable enzyme were isolated by shifting from a permissive (55 degrees C) to a nonpermissive (61 degrees C) temperature that inactivates the wild-type enzyme . DNA sequence analysis of the mutant genes revealed two types of mutation of single base substitution and hence a single amino acid replacement . The first type was the replacement of an aspartate by a tyrosine at position 80 of the wild-type enzyme, while the second was that of a threonine by a lysine at position 130 . Purified enzymes from the two mutant genes were confirmed to be substantially more thermostable than the wild type in vitro . The method of screening for a thermostable kanamycin nucleotidyltransferase presented here could be applied to any other enzyme, if a transformation system of a thermophile were available . Indeed, thermostable mutants with a subtle amino acid change would be of value for better understanding of forces and interactions that contribute to the stability of a protein.

Eur J Biochem, 1985 Dec 2, 153(2), 289 - 97
The primary structure of ribosomal protein L2 from Bacillus stearothermophilus; Kimura M et al.; The complete amino acid sequence of ribosomal protein L2 from the moderate thermophile Bacillus stearothermophilus has been determined . This has been achieved by the sequence analysis of peptides derived by enzymatic digestion with Staphylococcus aureus protease, trypsin and chymotrypsin, as well as by chemical cleavage with o-iodosobenzoic acid . The protein contains 275 amino acid residues and has a calculated molecular mass of 30201 Da . Comparison of this sequence with sequences of the corresponding proteins from Escherichia coli and from spinach and tobacco chloroplasts reveals that 60% of the residues of protein L2 from B . stearothermophilus are identical to those of the protein from E . coli and 45% are identical to those found in the two chloroplast proteins . There are extended regions of totally conserved sequence at positions 54-58 (GGGHK), 81-86 (EYDPNR), and 224-230 (MNPVDHP) in all four proteins.

Eur J Clin Microbiol, 1985 Dec, 4(6), 562 - 5
Serotyping of Campylobacter species by combined use of two methods; Jones DM et al.; Five hundred strains of thermophilic Campylobacter spp . from sporadic cases of enteritis and from epidemiologically related infections connected with outbreaks were serotyped . The haemagglutination method of Penner and the slide agglutination method of Lior were used together . Greater discrimination was obtained by the use of two methods together than by either alone; 96% of sporadic strains were typed using a restricted set of typing sera . Of the sporadic strains, 50% fell within five Penner serotypes and 50% fell within four Lior serotypes, so the increased discrimination obtained by using both methods was particularly useful amongst these most common serotypes . In outbreaks associated with one serotype both methods gave consistent results, and in outbreaks due to multiple serotypes the two methods complimented each other.

Biol Chem Hoppe Seyler, 1985 Dec, 366(12), 1079 - 83
Quinones from Archaebacteria, I . New types of menaquinones from the thermophilic archaebacterium Thermoproteus tenax; Thurl S et al.; From the archaebacterium Thermoproteus tenax, strain Kra-1 a mixture of 6 quinones of menaquinone and phylloquinone type with isopentyl side chains, MK-6(12H), MK-6-(10H), MK-5(10H), MK-5(8H), MK-4(8H), MK-4(6H) and two analogous quinones, containing in addition a methyl group in the naphthoquinone system, were isolated and characterized.

Proc Natl Acad Sci U S A, 1985 Dec, 82(23), 8048 - 52
Deposition-related histone acetylation in micronuclei of conjugating Tetrahymena; Allis CD et al.; Macro- and micronuclei of the ciliated protozoan, Tetrahymena thermophila, afford a unique opportunity to study histone acetylation under conditions where acetylation associated with the regulation of transcription and acetylation associated with the deposition of histones on the DNA are separable . In this study we demonstrate that histone H3 and histone H4 synthesized in young (5 hr) conjugating Tetrahymena are deposited into micronuclei in acetylated forms . Most of the newly synthesized histone H3 migrates as a monoacetylated form while essentially all of the new histone H4 is deposited as a diacetylated species . Since micronuclei replicate rapidly during this stage of the life cycle, but are transcriptionally inactive, these data suggest that histone acetylation is related functionally to histone deposition and chromatin assembly . Pulse-chase experiments show that micronuclei also contain a butyrate-sensitive deacetylase activity(ies) which operates to remove the deposition-related acetate groups from newly synthesized and deposited H3 and H4 . This enzymatic activity probably contributes to the steady state level of micronuclear histone acetylation that is low or nonexistent . Thus, evidence is emerging for at least two independent systems of histone acetylation in Tetrahymena . The first system is specific to macronuclei and may be related to gene expression . The second system is common to macro- or micronuclear histones (H3 and H4) and may be related to histone deposition during DNA replication.

Cell, 1985 Dec, 43(3 Pt 2), 747 - 58
The internally located telomeric sequences in the germ-line chromosomes of Tetrahymena are at the ends of transposon-like elements; Cherry JM et al.; The germ-line micronuclear genome of the ciliate Tetrahymena thermophila contains approximately 10(2) chromosome-internal blocks of tandemly repeated C4A2 sequences (mic C4A2) . This repeated sequence is the telomeric sequence in the somatic macronucleus . Each of six cloned micC4A2 was found to be adjacent to a conserved 30 bp sequence, which we propose is the terminal inverted repeat of a family of DNA elements (the Tel-1 family) . This 30 bp sequence contains a site for the infrequently cutting restriction enzyme Bst XI, which allows full-length Tel-1 elements to be cut out of the micronuclear genome . BAL 31 exonuclease digestion of Bst XI-cut micronuclear DNA showed the majority of micC4A2 blocks to be associated with the ends of the Tel-1 family . We propose that Tel-1 elements are transposable and suggest a novel mechanism to account for the origin of micC4A2, in which telomeric repeats are added to the ends of free linear forms of the transposable elements prior to reintegration.

J Bacteriol, 1985 Dec, 164(3), 1182 - 7
Efficient synthesis and secretion of a thermophilic alpha-amylase by protein-producing Bacillus brevis 47 carrying the Bacillus stearothermophilus amylase gene; Tsukagoshi N et al.; Bacillus subtilis and Bacillus brevis 47-5, carrying the Bacillus stearothermophilus alpha-amylase gene on pUB110 (pBAM101), synthesized the same alpha-amylase as the donor strain as determined by the enzyme's thermal stability and NH2-terminal amino acid sequence . Regardless of the host, the 34-amino acid signal peptide of the enzyme was processed at exactly the same site between two alanine residues . B . brevis 47-5(pBAM101) secreted the enzyme most efficiently of the hosts examined, 100, 15, and 5 times more than B . stearothermophilus, Escherichia coli HB101(pH1301), and B . subtilis 1A289(pBAM101), respectively . The efficient secretion of the enzyme in B . brevis 47-5(pBAM101) was suggested to be due to the unique properties of the cell wall of this organism.

Mol Biochem Parasitol, 1985 Dec, 17(3), 331 - 41
Comparative structural analysis of calmodulins from Trypanosoma brucei, T . congolense, T . vivax, Tetrahymena thermophila and bovine brain; Ruben L et al.; Calmodulin is an intracellular calcium receptor protein utilized extensively by eukaryotic cells to mediate responsiveness to calcium signals . The present study evaluates the effects on protein structure of amino acid substitutions in trypanosome calmodulin . Calmodulin conformation, hydrophobicity and antigenic determinants are compared among Trypanosoma brucei, Trypanosoma congolense, Trypanosoma vivax, Tetrahymena thermophila and bovine brain . Trypanosome calmodulin differs from brain and Tetrahymena calmodulins based upon isoelectric point, retention time on a C-2/C-18 reverse phase column and interaction with polyclonal antibodies against trypanosome calmodulin by radioimmunoassay or Western procedures . These same analyses do not distinguish trypanosome calmodulins from each other . Polyclonal antibodies against Tetrahymena calmodulin are equally specific and do not recognize the trypanosome or brain calmodulins . Calcium-induced exposure of hydrophobic binding sites are quantitated using the fluorescent probe, N-phenyl-1-naphthylamine . All calmodulins, regardless of source, enhance the fluorescence of N-phenyl-1-naphthylamine 3-4 fold in the presence of calcium . These data demonstrate the extent to which functional calmodulins vary in their structures . We conclude that African trypanosomes share a common calmodulin that is structurally distinct from calmodulin of vertebrates or Tetrahymena.

J Mol Biol, 1985 Nov 20, 186(2), 481 - 2
Preliminary X-ray diffraction studies on a ferredoxin from the thermophilic archaebacterium, Thermoplasma acidophilum; Tsukihara T et al.; A ferredoxin from the thermophilic archaebacterium, Thermoplasma acidophilum, is supposed to contain two (4Fe-4S) active centers; one center could be linked by four cysteine residues to the protein and the other bonded with three cysteines and an unknown group . This ferredoxin has been crystallized by salting-out against 2.3 M-ammonium sulfate solution . The space group is P21212 with cell dimensions of a = 59.20 A, b = 52.77 A and c = 41.28 A . Four molecules pack in the unit cell with Vm = 2.03 A3/dalton.

Nucleic Acids Res, 1985 Nov 11, 13(21), 7759 - 79
Self-catalyzed cyclization of the intervening sequence RNA of Tetrahymena: inhibition by methidiumpropyl.EDTA and localization of the major dye binding sites; Tanner NK et al.; The intervening sequence (IVS) excised from the rRNA precursor of Tetrahymena thermophila is converted to a covalently closed circular RNA in the absence of proteins in vitro . This self-catalyzed cyclization reaction is inhibited by the intercalating dye methidiumpropyl.EDTA (MPE; R.P . Hertzberg and P.B . Dervan (1982) J . Am . Chem . Soc . 104, 313-315) . The MPE binding sites have been localized by mapping the sites of MPE.Fe(II) cleavage of the IVS RNA . There are three major binding sites within the 414 nucleotide IVS RNA . Two of these sites coincide with the A.B and 9L.2 pairings . These are structural elements that are conserved in all group I introns and are implicated as being functionally important for splicing . We propose that interaction of MPE with these sites is responsible for dye inhibition of cyclization . The reactions of MPE.Fe(II) with an RNA of known structure, tRNAPhe, and with the IVS RNA were studied as a function of temperature, ionic strength and ethidium concentration . Based on the comparison of the reaction with these two RNAs, we conclude that the dye is a very useful probe for structural regions of large RNAs, while it provides more limited structural information about the small, compact tRNA molecule.

Antimicrob Agents Chemother, 1985 Nov, 28(5), 695 - 7
Susceptibility of Campylobacter jejuni and Campylobacter coli to macrolides and related compounds; Elharrif Z et al.; The susceptibility of 105 thermophilic campylobacters from human and swine origins to eight macrolides and related compounds was tested . Erythromycin, josamycin, clindamycin, and ASE 136 BS (a new erythromycin derivative) were the most active against the human strains . The swine strains were highly resistant, except to pristinamycin . The human Campylobacter coli strains (except for two strains) behaved like the C . jejuni strains.

J Protozool, 1985 Nov, 32(4), 640 - 4
The 5S and 5.8S ribosomal RNA sequences of Tetrahymena thermophila and T . pyriformis; Van Bell CT; The nucleotide sequences of the 5S rRNAs of Tetrahymena thermophila and two strains of T . pyriformis have been determined to be identical . The 5.8S rRNA sequences have also been determined; these sequences correct several errors in an earlier report . The 5.8S rRNAs of the two species differ at a single position . The sequencing results indicate that the species are of recent common ancestry . Molecular evidence that has been interpreted in the past as suggestive of an ancient divergence has been reviewed and found to be consistent with a T . pyriformis complex radiation beginning approximately 30-40 million years ago.

J Clin Pathol, 1985 Nov, 38(11), 1289 - 92
Rapid identification of thermophilic Naegleria, including Naegleria fowleri using API ZYM system; Kilvington S et al.; The suitability of the API ZYM system for identifying thermophilic Naegleria species, based on enzyme presence and activity, was investigated . Replicate testing on strains of N fowleri, N lovaniensis, and N australiensis cultured in a monoxenic and an axenic medium showed that the system could provide a rapid and reproducible means of identifying the species soon after primary isolation . No single enzyme was found specific for any one species, but considerable differences were found in the patterns of activity of acid phosphatase and leucine arylamidase . When these were compared the species could be differentiated . Use of the system in conjunction with a simple culture method is proposed as a readily available means of monitoring environmental and public bathing sites to prevent primary amoebic meningoencephalitis.

J Clin Microbiol, 1985 Nov, 22(5), 714 - 8
Comparison of four hippurate hydrolysis methods for identification of thermophilic Campylobacter spp; Morris GK et al.; The test for hippurate hydrolysis is critical for separation of Campylobacter jejuni and C . coli strains . Glycine and benzoic acid are formed when hippurate is hydrolyzed by C . jejuni . The test used in most laboratories is one of several variations of the ninhydrin tube test described by Hwang and Ederer (M . Hwang and G . M . Ederer, J . Clin . Microbiol . 1:114-115, 1975) for detection of glycine . We evaluated three modifications of the Hwang and Ederer method and the gas-liquid chromatographic (GLC) method described by Kodaka et al . (H . Kodaka, G . L . Lombard, and V . R . Dowell, Jr., J . Clin . Microbiol . 16:962-964, 1982) for detecting benzoic acid . Campylobacter strains comprised 22 C . jejuni, 11 C . coli, and 8 C . laridis strains . The species identification of each strain was confirmed by DNA relatedness . All strains of C . jejuni were positive and all strains of C . coli and C . laridis were negative by the GLC method for detecting hippurate hydrolysis, whereas three strains of C . jejuni gave negative or variable results in the tube tests . The GLC method is more sensitive than the tube methods for detecting hippurate hydrolysis and should be used on cultures yielding variable or questionable test results.

Can J Microbiol, 1985 Nov, 31(11), 1006 - 10
Oxygen defense systems in obligately thermophilic bacteria; Allgood GS et al.; Ten strains of Gram-negative, aerobic, obligately thermophilic bacteria were examined for their response to oxygen toxicity by comparing static with shaken cultures . All of the organisms tested had measurable levels of superoxide dismutase, catalase, and peroxidase . Aeration generally did not result in an increased level of superoxide dismutase in any of the thermophiles . Aeration of organisms obligate for n-alkane substrate caused an increase in cellular peroxidase levels and a corresponding decrease in catalase . The thermophiles that grew on either n-alkanes or complex media did not grow on the hydrocarbon in aerated culture but on a complex medium, aeration effected an increased level of catalase . With the exception of a pink-pigmented thermophile which, when aerated, did not have an increased level of the three oxygen defense enzymes, most of the thermophiles surveyed had an increased level of catalase or peroxidase when exposed to increased oxygen tension . The activity of the enzymes was determined at temperatures from 25 to 65 degrees C and the former temperature was satisfactory for these experiments.

J Bacteriol, 1985 Nov, 164(2), 674 - 83
Comparative stability and catalytic and chemical properties of the sulfate-activating enzymes from Penicillium chrysogenum (mesophile) and Penicillium duponti (thermophile); Renosto F et al.; ATP sulfurylases from Penicillium chrysogenum (a mesophile) and from Penicillium duponti (a thermophile) had a native molecular weight of about 440,000 and a subunit molecular weight of about 69,000 . (The P . duponti subunit appeared to be a little smaller than the P . chrysogenum subunit.) The P . duponti enzyme was about 100 times more heat stable than the P . chrysogenum enzyme; k inact (the first-order rate constant for inactivation) at 65 degrees C = 3.3 X 10(-4) s-1 for P . duponti and 3.0 X 10(-2) s-1 for P . chrysogenum . The P . duponti enzyme was also more stable to low pH and urea at 30 degrees C . Rabbit serum antibodies to each enzyme showed heterologous cross-reaction . Amino acid analyses disclosed no major compositional differences between the two enzymes . The analogous Km and Ki values of the forward and reverse reactions were also essentially identical at 30 degrees C . At 30 degrees C, the physiologically important adenosine 5'-phosphosulfate (APS) synthesis activity of the P . duponti enzyme was 4 U mg of protein-1, which is about half that of the P . chrysogenum enzyme . The molybdolysis and ATP synthesis activities of the P . duponti enzyme at 30 degrees C were similar to those of the P . chrysogenum enzyme . At 50 degrees C, the APS synthesis activity of the P . duponti enzyme was 12 to 19 U mg of protein-1, which was higher than that of the P . chrysogenum enzyme at 30 degrees C (8 +/- 1 U mg of protein-1) . Treatment of the P . chrysogenum enzyme with 5,5'-dithiobis(2-nitrobenzoate) (DTNB) at 30 degrees C under nondenaturing conditions modified one free sulfhydryl group per subunit . Vmax was not significantly altered, but the catalytic activity at low magnesium-ATP or SO4(2-) (or MoO4(2-)) was markedly reduced . Chemical modification with tetranitromethane had the same results on the kinetics . The native P . duponti enzyme was relatively unreactive toward DTNB or tetranitromethane at 30 degrees C and pH 8.0 or pH 9.0, but at 50 degrees C and pH 8.0, DTNB rapidly modified one SH group per subunit . APS kinase (the second sulfate-activating enzyme) of P . chrysogenum dissociated into inactive subunits at 42 degrees C . The P . duponti enzyme remained intact and active at 42 degrees C.

J Protozool, 1985 Nov, 32(4), 644 - 9
RNA and protein synthesis during meiotic prophase in Tetrahymena thermophila; Martindale DW et al.; Tetrahymena is one of the few organisms from which large amounts of precisely staged meiotic material can be obtained . We took advantage of this fact to monitor RNA and protein synthesis during meiosis . The rate of total protein synthesis as well as the synthesis of the majority of heavily labeled conjugation-specific polypeptides (monitored by high resolution two-dimensional gel electrophoresis) was maximal during meiotic prophase . We therefore cloned cDNAs corresponding to genes active during this time . The mRNA levels of three conjugation-specific genes (pC1, pC2, and pC7) and one conjugation-induced gene (pC3) were followed by using the corresponding labeled cDNAs to probe RNA isolated from different times during mating that was also followed cytologically . Synthesis of the conjugation-specific mRNAs was maximal just prior to maximum crescent stage (pachytene) . Evidence is presented for transcription by the normally inactive micronucleus just prior to the maximum crescent stage, confirming an earlier report . The significance of these results is discussed.

J Bacteriol, 1985 Nov, 164(2), 878 - 81
Apurinic and apyrimidinic DNA endonuclease of extremely thermophilic Thermothrix thiopara; Kaboev OK et al.; An endonuclease specific for apurinic, apyrimidinic (AP) sites in DNA was purified nearly to homogeneity from the extremely thermophilic bacterium Thermothrix thiopara . The enzyme has a molecular weight of approximately 26,000 . It cleaves neither native nor UV- or gamma-irradiated DNAs and has no contaminating exonuclease or uracil-DNA glycosylase activities . The enzyme has no cofactor requirement and is not inhibited by EDTA or N'-ethylmaleimide . It shows maximal activity at 70 degrees C and a pH between 7.5 and 9.0 . The Arrhenius activation energy of the reaction is 17 kJ/mol, and the apparent Km for AP sites is 38 nM . The rate of heat inactivation of the enzyme followed first-order kinetics, with a half-life of 10 min at 70 degrees C but about 150 min in the presence of 0.5 M ammonium sulfate or 0.5 mg of bovine serum albumin per ml at the same temperature . One cell of T . thiopara contains sufficient AP endonuclease activity for hydrolysis of about 10(6) phosphodiester bonds per h at 70 degrees C . An extract of these bacteria does not contain detectable Mg-dependent AP endonuclease activity, and the above-mentioned enzyme appears to be the main AP endonuclease of T . thiopara.

Biochemistry, 1985 Oct 8, 24(21), 5711 - 5
Two tRNAIle1 species from an extreme thermophile, Thermus thermophilus HB8: effect of 2-thiolation of ribothymidine on the thermostability of tRNA; Horie N et al.; From Thermus thermophilus HB8 grown at 65 degrees C, two major tRNAIle species have been purified by column chromatography and polyacrylamide gel electrophoresis . The nucleotide sequence of one of these two tRNAIle1 species (tRNAIle1a) has been determined to be pGGGCGAUUAGCUCAGCUGmGUDAGAGCGCACGCCUGAUt6AAGCGUGAGm7GUCGGUGGs2T psi CAm1AGUCCACCAUCGCCCACCAOH . The nucleotide sequence of the other species (tRNAIle1b) is found to be the same as that of tRNAIle1a except for the modification in position 54; tRNAIle1a has s2T(54) while tRNAIle1b has T(54) . The melting temperature of tRNAIle1a is as high as 86.2 degrees C while that of tRNAIle1b is 83.3 degrees C . The single replacement of an oxygen atom (2-carbonyl oxygen) of T(54) by a sulfur atom significantly contributes to the thermostability of the tRNAIle1a species . In addition, the methylation of G(18) and A(58) possibly contributes to the thermostability of T . thermophilus tRNAIle1a and tRNAIle1b species.

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