EMBO J, 1994 Dec 1, 13(23), 5721 - 31 Architecture of telomerase RNA; Bhattacharyya A et al.; Telomerase, an essential ribonucleoprotein reverse transcriptase, adds telomeric DNA to the ends of eukaryotic chromosomes . We examined the conformational properties of the naked RNA moiety of telomerase from two related ciliates, Tetrahymena thermophila and Glaucoma chattoni . As well as finding evidence for features proposed previously on the basis of phylogenetic comparisons, novel conserved structural properties were revealed . Specifically, although the region around helix III was previously proposed to form a pseudoknot, our results indicate that in the naked RNA this region maintains a level of 'plasticity', probably in an equilibrium favoring one of two helices . In addition, these studies reveal that the templating domain is not entirely single-stranded as previously proposed, but is ordered due to constraints imposed by other parts of the RNA . Finally, our results suggest that the GA bulge in helix IV may introduce a structurally conserved kink . We now propose a 'two-domain' structure for the telomerase RNA based on function: one conformationally flexible domain, which includes the template and the region around helix III, involved with enzymatic function, and a second largely helical domain, including helices I and IV and the proposed kink, which may serve as a scaffold for protein binding. EMBO J, 1994 Dec 1, 13(23), 5517 - 22 Protein splicing: an analysis of the branched intermediate and its resolution by succinimide formation; Xu MQ et al.; Protein splicing involves the excision of an internal domain from a precursor protein and the ligation of the external domains so as to generate two new proteins . Study of this process has recently been facilitated by the isolation of a precursor and a branched intermediate from a thermophilic protein splicing element expressed in a foreign protein context . Two aspects of protein splicing are examined in this paper . We demonstrate a succinimide at the C-terminus of the spliced internal protein, implicating cyclization of asparagine in resolution of the branched intermediate, and we identify an alkali-labile bond in the branched intermediate . A revised protein splicing model based on these experimental results is presented. Arch Biochem Biophys, 1994 Dec, 315(2), 262 - 6 Characterization of flavocytochrome C552 from the thermophilic photosynthetic bacterium Chromatium tepidum; Garcia Castillo MC et al.; A M(r) 68 kDa flavocytochrome c552 has been isolated from the thermophilic photosynthetic purple sulfur bacterium Chromatium tepidum and shown to consist of a M(r) 25 kDa subunit that contains two covalently bound heme c and a M(r) 43 kDa subunit that probably contains a single FAD . The prosthetic group content, absorbance spectra, and subunit composition of the C . tepidum flavocytochrome are quite similar to those previously reported for the flavocytochrome c552 isolated from a mesophilic Chromatium species, Chromatium vinosum . The oxidation-reduction properties of the hemes present in the C . tepidum flavocytochrome have been characterized by titrations, the effect of temperature on the catalytic activity of the protein has been investigated, and the heme environment has been characterized using resonance Raman spectroscopy. Nature, 1994 Dec 1, 372(6505), 455 - 8 Anaerobic oxidation of hydrocarbons in crude oil by new types of sulphate-reducing bacteria; Rueter P et al.; Many crude oil constituents are biodegradable in the presence of oxygen; however, a substantial anaerobic degradation has never been demonstrated . An unusually low content of n-alkanes in oils of certain deposits is commonly attributed to selective utilization of these hydrocarbons by aerobic microorganisms . On the other hand, oil wells and production fluids were shown to harbour anaerobic sulphate-reducing bacteria, but their actual electron donors and carbon sources were unknown . On the basis of nutritional properties of various bacterial isolates it was assumed that fatty acids and H2 are potential electron donors for sulphate reduction in situ . Here we demonstrate that hydrocarbons in crude oil are used directly by sulphate-reducing bacteria growing under strictly anoxic conditions . A moderately thermophilic pure culture selectively utilizes n-alkanes in oil for sulphate reduction to sulphide . In addition, a mesophilic sulphate-reducing enrichment culture is shown to oxidize alkylbenzenes in oil . Thus, sulphate-reducing bacteria utilizing aliphatic and aromatic hydrocarbons as electron donors may present a significant source of sulphide in oil deposits and oil production plants. J Bacteriol, 1994 Dec, 176(23), 7387 - 90 Characterization of the Thermus thermophilus locus encoding peptide deformylase and methionyl-tRNA(fMet) formyltransferase; Meinnel T et al.; An Escherichia coli strain with thermosensitive expression of the gene encoding peptide deformylase (fms) has been constructed . At nonpermissive temperatures, this strain fails to grow . The essential character of the fms gene was further used to clone by heterologous complementation the locus corresponding to Thermus thermophilus peptide deformylase . The cloned fragment also carries the methionyl-tRNA(fMet) formyltransferase gene (fmt) . It is located immediately downstream from the fms gene, as in E . coli . Further sequence analysis of the region surrounding the E . coli fms-fmt locus indicates that the genes bordering the fms-fmt region are not conserved in T . thermophilus. J UOEH, 1994 Dec 1, 16(4), 263 - 75 Intracellular multiplication of Legionella pneumophila in Tetrahymena thermophila; Kikuhara H et al.; It has been shown that Legionella pneumophila proliferates intracellularly in more than ten species of protozoa, but the fate of the bacteria in Tetrahymena thermophila has not been reported . We investigated the multiplication of L . pneumophila Philadelphia-1 strain in micronucleated T . thermophila, and the effects of temperature and numbers of the bacteria ingested by the protozoa after in vitro feeding were studied . T . thermophila preyed actively upon the bacteria . After being ingested, the fate of the bacteria was affected by both temperature and the number of bacteria ingested . When the number of ingested bacteria was 30 per protozoon, the bacteria proliferated intracellularly at 35 degrees C . The bacteria, however, could not proliferate at 28 degrees C or 32 degrees C though they survived in the protozoa . When the ingested bacteria was 10 per protozoon, the bacteria were killed in the protozoa at all of the temperatures tested . Electron microscopic examination revealed that the protozoa ingesting the bacteria remarkably swelled and that protozoan food vacuoles which contained L . pneumophila were studded with ribosomes. Appl Environ Microbiol, 1994 Dec, 60(12), 4537 - 43 Detection and classification of Streptococcus thermophilus bacteriophages isolated from industrial milk fermentation; Brussow H et al.; In the last 30 years, 81 Streptococcus thermophilus bacteriophage isolates were collected from industrial yogurt (n = 40) and cheese (n = 41) fermentation . Forty-six distinct restriction patterns of phage DNA (11 in yogurt and 35 in cheese) were observed . The phages were investigated for host range, serological properties, and DNA homology to study whether these three independent techniques can be used to classify the phages into taxonomic groups . Yogurt factory-derived phages were classified into the same two subgroups by serology, host range analysis, and hybridization with subgroup-specific DNA sequences . Cheese factory-derived phages, however, could not be classified: the 35 cheese phage isolates with distinct restriction patterns showed 34 different host ranges . All but one cheese phage isolate showed serological cross-reactivity with yogurt phages . A phage DNA fragment that hybridized with all phage DNA samples was cloned, establishing the genetic relatedness of all S . thermophilus phages from our collection . With the sequence information from an unusually conserved S . thermophilus phage DNA element (H . Brussow, A . Probst, M . Fremont, and J . Sidoti, Virology 200:854-857, 1994), a PCR-based phage detection method was developed for cheese whey from a factory that produced mozzarella cheese with complex undefined starter mixes . PCR allowed the detection of phages in cheese whey (detection limit, 10(3) PFU/ml) which could not be detected by dot blot hybridization techniques (detection limit, 10(7) PFU/ml). Appl Microbiol Biotechnol, 1994 Dec, 42(4), 569 - 74 Fermentation conditions for efficient production of thermophilic protease in Escherichia coli harboring a plasmid; Sakamoto S et al.; Escherichia coli TG1, transformed with an expression plasmid pAQN carrying the aqualysin I (AQI) gene derived from Thermus aquaticus YT-1 under the control of the tac promoter, was cultivated under various conditions in order to find fermentation conditions for the efficient production of the thermophilic protease, AQI . The amount of AQI produced was closely related to the growth phase at the time of isopropyl-beta-D-thiogalactopyranoside (IPTG) induction, and the highest production was obtained when it was added during the exponential growth phase . The addition of yeast extract had a greater effect on AQI production than did Polypeptone or casamino acids, and AQI productivity increased from 1.1 x 10(3) kU/g to 2.7 x 10(3) kU/g cells when 2 g/l yeast extract was supplied . Furthermore, the specific growth rate improved from 0.35 h-1 to 0.89 h-1 when 5 g/l yeast extract was supplied . The culture temperature also affected AQI gene expression . When the temperature was shifted from 37 degrees C to 34 degrees C at the time of IPTG induction, 19 kU/ml enzymatically active AQI was obtained, corresponding to a 28% increase over the amount produced in a batch culture without a shift . This is about a 44-fold higher yield than was obtained from the original strain, T . aquaticus YT-1. Protein Sci, 1994 Dec, 3(12), 2340 - 50 Conserved sequence features of inteins (protein introns) and their use in identifying new inteins and related proteins; Pietrokovski S; Inteins (protein introns) are internal portions of protein sequences that are posttranslationally excised while the flanking regions are spliced together, making an additional protein product . Inteins have been found in a number of homologous genes in yeast, mycobacteria, and extreme thermophile archaebacteria . The inteins are probably multifunctional, autocatalyzing their own splicing, and some were also shown to be DNA endonucleases . The splice junction regions and two regions similar to homing endonucleases were thought to be the only common sequence features of inteins . This work analyzed all published intein sequences with recently developed methods for detecting weak, conserved sequence features . The methods complemented each other in the identification and assessment of several patterns characterizing the intein sequences . New intein conserved features are discovered and the known ones are quantitatively described and localized . The general sequence description of all the known inteins is derived from the motifs and their relative positions . The intein sequence description is used to search the sequence databases for intein-like proteins . A sequence region in a mycobacterial open reading frame possessing all of the intein motifs and absent from sequences homologous to both of its flanking sequences is identified as an intein . A newly discovered putative intein in red algae chloroplasts is found not to contain the endonuclease motifs present in all other inteins . The yeast HO endonuclease is found to have an overall intein-like structure and a few viral polyprotein cleavage sites are found to be significantly similar to the inteins amino-end splice junction motif . The intein features described may serve for detection of intein sequences. Biol Pharm Bull, 1994 Dec, 17(12), 1543 - 8 Studies on thermophile products . XI . Biological effect of antigen presenting inhibitor, isofatty acid-containing phosphatidylethanolamine, on mouse macrophages; Kohama Y et al.; A new fraction, Fr . 8-A, which consists of a phosphatidylethanolamine with C14:0-C18:0 isofatty acids was obtained from Bacillus stearothermophilus UBT8038 . The fraction inhibited major histocompatibility complex class II (Ia) antigen expression and antigen presentation on mouse macrophages . The effect of Fr.8-A on macrophage functions related to antigen presentation was investigated . Fr . 8-A increased arachidonate release, prostaglandin (PG) E2 release and nitrite production from peritoneal macrophages . It increased further the levels of PGE2, nitrite and tumor necrosis factor in the culture supernatant of the macrophages induced by the supernatant from concanavalin A-stimulated spleen cell cultures . Fr . 8-A augmented the activity of peritoneal macrophages to suppress Con A-stimulated T cell proliferation . Addition of either indomethacin or Ng-methyl-L-arginine had no effect on the augmentation of suppressor macrophage activity or the inhibition of antigen presentation by Fr . 8-A, while simultaneous addition of both inhibitors abrogated the effect of the fraction . These results indicate that Fr . 8-A inhibits Ia expression and antigen presentation, and augments suppressor macrophage activity at least partly via the activation of both cyclooxygenase and nitric oxide synthase pathways. Protein Eng, 1994 Dec, 7(12), 1471 - 8 The effect of ion pairs on the thermal stability of D-glyceraldehyde 3-phosphate dehydrogenase from the hyperthermophilic bacterium Thermotoga maritima; Tomschy A et al.; D-Glyceraldehyde 3-phosphate dehydrogenase from Thermotoga maritima (TmGAPDH) is intrinsically thermostable, exhibiting a thermal transition beyond 105 degrees C . Neither the amino-acid composition nor homology modelling, based on sequence alignment and known 3-D structures of the enzyme from meso- and thermophiles, provide an explanation of the anomalous stability . Recent X-ray data suggest that an increased number of ion pairs is involved . To prove this hypothesis, a number of charged residues contributing to ion pairs in TmGAPDH, but absent in the moderately thermophilic enzyme were altered . Elimination of peripheral ion pairs (E103-K104, E261-R266) was found to be ineffective . Altering a central charge cluster (R10-D47, E314, D*186) led to a drastic decrease in coenzyme binding . As a consequence, guanidine-dependent deactivation is shifted to significantly lower guanidinium chloride (GdmCl) concentrations without altering the denaturation/dissociation profile of the wild type enzyme . Mutants in the S loop (R195D, R195D-D181K) lead to a biphasic profile in the GdmCl-dependent denaturation transition and significant destabilization; at room temperature no subunit dissociation could be observed. J Dairy Sci, 1994 Dec, 77(12), 3532 - 7 Influence of bile on beta-galactosidase activity of component species of yogurt starter cultures; Noh DO et al.; The influence of bile on beta-galactosidase activity and the cellular integrity of Lactobacillus delbrueckii ssp . bulgaricus and Streptococcus salivarius ssp . thermophilus were tested . Two strains (OS 1 and OS 2) of L . delbrueckii ssp . bulgaricus and three strains (HC 15, 17, and 18) of S . salivarius ssp . thermophilus were studied . In the presence of .15% oxgall, beta-galactosidase activity of whole cells of both species was significantly increased . However, at a higher concentration (.3% oxgall), the beta-galactosidase activity was significantly less than in the presence of .15% oxgall . The presence of oxgall did not promote the cell lysis of any of the strains of either species . Additionally, the presence of oxgall did not cause the leakage of the enzyme from the cells . Thus, the presence of oxgall increased the cellular permeability to allow more substrate to enter the cells, thereby increasing beta-galactosidase activity, and, at higher concentrations, was inhibitory to beta-galactosidase activity of both species. J Vet Med Sci, 1994 Dec, 56(6), 1129 - 33 Enhancement of cytotoxic activity of lymphocytes in mice by oral administration of peptidoglycan (PG) derived from Bifidobacterium thermophilum; Sasaki T et al.; A preparation of peptidoglycan (PG) of Bifidobacterium thermophilum (B . thermophilum) of swine was orally administered to SPF-C57BL/6CrSlc mice in order to confirm the enhancement of the cytotoxic activity of natural killer cells (NK), intraperitoneal cytotoxic T lymphocytes (CTL) and lymphocytes stimulated by concanavalin A (Con A-stimulated lymphocytes) . The NK cells from the spleen and the mesenteric lymph node (MLN) of mice that were continuously fed with PG-mixed feed for three weeks showed a significantly higher rate of cytolysis than those from the control group . However, a single oral administration of PG had no significant effect on NK activity . The activity of peritoneally sensitized CTL of the mice that were continuously fed with PG-mixed feed was assayed . The PG-mixed feed administered group showed a higher CTL activity than that of the control group . The cytotoxic activity of Con A-stimulated lymphocytes in the PG-mixed feed administered group was higher than that of the control group . These results indicate that the cytotoxic activity of mice was enhanced by orally administered PG. Mycopathologia, 1994 Dec, 128(3), 139 - 41 Extracellular amylase activities of Rhizomucor pusillus and Humicola lanuginosa at initial stages of growth; Adams PR; Among thermophilic fungi, Rhizomucor Pusillus and Humicola lanuginosa have been reported to be among the most prolific producers of amylase, an apparently heat stable enzyme vital to the incorporation of carbon from macromolecular sources such as starch . Yet the highest levels of extracellular amylase in starch-yeast cultures of these fungi were measured after most of the growth had occurred; pre-growth levels appeared to be very small . Since these low levels are the significant ones for growth, a procedure was devised to measure them: 1.162 x 10(-2) units (mg maltose/ml/min) were measured after two days of growth of R . pusillus and 6.230 x 10(-3) units measured after four days of the slower-growing H . lanuginosa . Re-assays of these after dialysis to remove most of the reducing sugars gave 1.689 x 10(-2) units and 1.234 x 10(-2) units, respectively, with all correlation coefficients 0.96 or better. Microbiology, 1994 Dec, 140 ( Pt 12), 3451 - 6 Characterization of Thiobacillus caldus sp . nov., a moderately thermophilic acidophile; Hallberg KB et al.; Two isolates of a novel, moderately thermophilic Thiobacillus species have been studied . The isolates, KU and BC13, are Gram-negative, motile bacteria having a pH optimum for growth of 2-2.5 and an optimum growth temperature of 45 degrees C . Both isolates are capable of chemolithotrophic growth on reduced sulfur substrates . They can also use molecular hydrogen as an electron donor . These two isolates can grow mixotrophically with sulfur or tetrathionate and yeast extract or glucose . The G+C content is 63.1-63.9 mol% and the isolates exhibit no significant DNA homology to any other Thiobacillus species . Strains KU and BC13 both contain ubiquinone Q-8 . 16S rRNA analysis indicates that these strains belong to a group of bacteria which includes other chemolithotrophic sulfur oxidizers such as T . ferrooxidans and T . thiooxidans . These characteristics distinguish KU and BC13 from any other species described previously and they thus represent the first acidophilic, thermophilic Thiobacillus species, named T . caldus sp . nov., to be described . The type strain, referred to as strain KU in this paper, has been deposited in the Deutsche Sammlung von Mikroorganismen, Braunschweig, FRG, with the accession number DSM 8584. FEBS Lett, 1994 Nov 28, 355(2), 171 - 2 Roles of Arg231 and Tyr284 of Thermus thermophilus isocitrate dehydrogenase in the coenzyme specificity; Yaoi T et al.; The coenzyme binding site of isocitrate dehydrogenase from Thermus thermophilus was analyzed by site-directed mutagenesis . The mutation analysis revealed that Arg231 and Tyr284 are involved in the discrimination between NAD and NADP, suggesting that these two residues interact with 2'-phosphate group of NADP. J Mol Biol, 1994 Nov 25, 244(2), 242 - 9 Structure and transcription of the L11-L1-L10-L12 ribosomal protein gene operon from the extreme thermophilic archaeon Sulfolobus acidocaldarius; Ramirez C et al.; We have cloned and sequenced four ribosomal protein genes from the extreme thermophilic archaeon Sulfolobus acidocaldarius P1 . These genes code for proteins equivalent to L11, L1, L10 and L12 from Escherichia coli . The genes for the Sulfolobus L11, L1, L10 and L12 proteins are arranged in the same order as the equivalent genes in E . coli, i.e . L11-L1-L10-L12, and are transcribed as a single unit . Sequences resembling the consensus sequence for archaeal promoters have been detected upstream of the transcription initiation site . Transcription ends at several sites following a pyrimidine-rich region . The genes for proteins L11, L10 and L1 start with unusual initiation codons: GUG in the case of the L1 and L10 genes; and UUG in the case of L11 . There are overlapping stop/start codons between the L11 and L1 genes, and between the L1 and L10, suggesting that the translation of the four genes might be coupled as in the bacteria. J Mol Biol, 1994 Nov 25, 244(2), 158 - 67 Synthesis and recognition of aspartyl-adenylate by Thermus thermophilus aspartyl-tRNA synthetase; Poterszman A et al.; The crystal structures of Thermus thermophilus aspartyl-tRNA synthetase and of its complex with ATP, Mg2+ and aspartic acid, show in situ formation of the amino acid adenylate and furnish experimental evidence for the modes of recognition of aspartic acid and ATP . The amino acid fits in a predefined specific site in which it replaces water molecules without significant conformational changes of the binding residues . This mode of selection is reminiscent of the lock and key concept . The pocket is closed by the movement of a histidine side chain from a neighbouring loop acting as a valve . ATP binding is driven by the stacking of the adenine upon the otherwise fixed aromatic ring of the class-II-invariant phenylalanine Phe235 . Specific recognition is achieved by interactions with the flexible side chains of other class-II-conserved residues . Conformational changes have been identified which allow the description of a reaction pathway including both lock-and-key and induced-fit interactions . This pathway can presumably be extended to all class II aaRS. Biochemistry, 1994 Nov 22, 33(46), 13864 - 79 Dissection of the role of the conserved G.U pair in group I RNA self-splicing; Knitt DS et al.; Phylogenetic conservation among > 100 group I introns and previous in vitro studies have implicated a G.U pair as defining the 5'-splice site for exon ligation . The U residue defines the 3' end of the 5' exon, and the complementary G residue is part of the internal guide sequence (IGS) that base pairs to the 5' exon . We now quantitate the effect of this pair on individual reaction steps using the L-21ScaI ribozyme, which is derived from the group I intron of Tetrahymena thermophila pre-rRNA . The following results indicate that interactions with this G.U pair contribute to the binding of the 5'-exon, the positioning of the 5'-splice site with respect to the catalytic site, and the chemical step . The oligonucleotide, CCCUCU, binds to the ribozyme approximately 20-fold stronger than CCCUCC despite the fact that the U-containing oligonucleotide forms an approximately 5-fold less stable duplex with an oligonucleotide analog of the IGS, GGAGGG . This and two independent experimental observations indicate that the G.U pair contributes approximately 100-fold (3 kcal/mol, 50 degrees C) to tertiary interactions that allow the P1 duplex, which is formed between the 5'-exon and the IGS, to dock into the ribozyme's core . The approximately 50-80-fold increase in miscleavage of 5'-exon analogs upon replacement of the 3'-terminal U of CCCUCU with C or upon removal of the 3'-terminal U suggests that the tertiary interactions with the G.U pair not only contribute to docking but also ensure correct positioning of the 5'-splice site with respect to the catalytic site, thereby minimizing the selection of incorrect splice sites . Comparison of the rates of the chemical cleavage step with G.U vs G.C suggests that the G.U pair contributes approximately 10-fold to the chemical step . It was previously suggested that the 2'-hydroxyl of this U residue helps stabilize the 3'-oxyanion leaving group in the chemical transition state via an intramolecular hydrogen bond . Relative reactivities of oligonucleotide substrates with ribose and deoxyribose U and C are consistent with a model based on a recent X-ray crystallographic structure in which the exocyclic amino group of G helps orient the 2'-hydroxyl of U via a bridging water molecule, thereby strengthening the hydrogen bond donated from the 2'-hydroxyl group to the neighboring incipient 3'-oxyanion . Finally, kinetic and thermodynamic evidence for the formation of a G.C+ wobble pair is presented.(ABSTRACT TRUNCATED AT 400 WORDS) J Biol Chem, 1994 Nov 18, 269(46), 28885 - 92 Purification and characterization of two forms of I-DmoI, a thermophilic site-specific endonuclease encoded by an archaeal intron; Dalgaard JZ et al.; The archaeal intron in the 23 S rRNA gene of the hyperthermophile Desulfurococcus mobilis has previously been shown to encode a site-specific DNA endonuclease that contains the LAGLIDADG motif . The enzyme, I-DmoI, has been shown to be active in two forms when expressed in vitro, from RNAs representing either the linear (I-DmoIl) or circular (I-DmoIc) intron . In this study we have overexpressed I-DmoIl and I-DmoIc and purified the enzymes from Escherichia coli . The optimal conditions for the enzymatic activity in vitro were determined, and the enzyme was used to delimit the recognition boundary on its DNA substrate (14-20 nucleotides), an intronless 23 S rRNA gene . Despite belonging to the archaeal kingdom, and being the product of a hyperthermophile, I-DmoI shares many properties with LAGLIDADG intron and intein endonucleases in other kingdoms . These results support the view that these phylogenetically diverse enzymes, which function to mobilize the DNA sequences that encode them, share a common ancestry. Eur J Biochem, 1994 Nov 15, 226(1), 169 - 77 Purification and characterization of the 30S ribosomal proteins from the bacterium Thermus thermophilus; Tsiboli P et al.; The total protein mixture of the 30S subunit (TP-30) of the bacterium Thermus thermophilus has been purified using reverse-phase HPLC and the proteins obtained were identified both by means of two-dimensional polyacrylamide gel electrophoresis as well as by amino-terminal amino acid microsequence analysis . The proteins are numbered according to their primary structural similarity with known prokaryotic ribosomal proteins . Eight of them, namely proteins S6, S7, S9, S10, S14, S15, S16 and S17 run at different positions in the two-dimensional gel electrophoresis system to those suggested {Sedelnikova, S . C., Agalarov, M . B., Garber, M . & Yusupov, M . M . (1987) FEBS Lett . 220, 227-230} . All characterized proteins are homologous to known ribosomal proteins from other species, except for a small basic protein which shows homology only to a ribosomal protein from spinach chloroplasts {Choli, T., Franceschi, F., Yonath, A . & Wittmann-Leibold, B . (1993) Biol . Chem . Hoppe-Seyler 374, 377-383; Subramanian, A.-R . (1984) Trends Biochem . Sci . 9, 491-494}. Structure, 1994 Nov 15, 2(11), 1007 - 16 Structure of 3-isopropylmalate dehydrogenase in complex with NAD+: ligand-induced loop closing and mechanism for cofactor specificity; Hurley JH et al.; BACKGROUND: The leucine biosynthetic enzyme 3-isopropylmalate dehydrogenase (IMDH) belongs to a unique class of bifunctional decarboxylating dehydrogenases . The two best-known members of this family, IMDH and isocitrate dehydrogenase (IDH), share a common structural framework and catalytic mechanism but have different substrate and cofactor specificities . IMDH is NAD(+)-dependent, while IDHs occur in both NAD(+)-dependent and NADP(+)-dependent forms . RESULTS: We have co-crystallized Thermus thermophilus IMDH with NAD+ and have determined the structure at 2.5 A resolution . NAD+ binds in an extended conformation . Comparisons with the structure in the absence of cofactor show that binding induces structural changes of up to 2.5 A in the five loops which form the dinucleotide-binding site . The adenine and diphosphate moieties of NAD+ are bound via interactions which are also present in the NADP(+)-IDH complex . Amino acids which interact with the NADP+ 2'-phosphate in IDH are substituted or absent in IMDH . The adenosine ribose forms two hydrogen bonds with Asp278, and the nicotinamide and nicotinamide ribose interact with Glu87 and Asp78, all unique to IMDH . CONCLUSIONS: NAD+ binding induces a conformational transition in IMDH, resulting in a structure that is intermediate between the most 'open' and 'closed' decarboxylating dehydrogenase conformations . Physiological specificity of IMDH for NAD+ versus NADP+ can be explained by the unique interaction between Asp278 and the free 2'-hydroxyl of the NAD+ adenosine, discrimination against the presence of the 2'-phosphate by the negative charge on Asp278, and the absence of potential favorable interactions with the 2'-phosphate of NADP+. J Mol Biol, 1994 Nov 4, 243(4), 806 - 8 Crystallization and preliminary X-ray crystallographic studies of thermostable xylanase crystals isolated from Paecilomyces varioti; Eswaramoorthy S et al.; A highly thermostable xylanase isolated from the thermophilic fungus Paecilomyces varioti has been crystallized by the vapour diffusion method . The isolation of this enzyme by crystallization directly from the culture filtrate projects this fungus as an important source for large-scale production of pure xylanase . The crystals belong to orthorhombic space group P2(1)2(1)2(1) with the unit cell dimensions a = 38.48 A, b = 53.87 A and c = 90.23 A . Four molecules occupy a volume of 187,039.4 A3 along with 34% of solvent . The data collected with an area detector to the resolution of 2.7 A were used to calculate the unit cell parameters and Matthews' constant . The optical behaviour of the crystal was studied at different temperatures to understand its thermal stability. Gene, 1994 Nov 4, 149(1), 127 - 36 Trapping DNA polymerases using triplex-forming oligodeoxyribonucleotides; Samadashwily GM et al.; Triplexes (triple helices) formed within DNA templates prior to or during DNA synthesis cause DNA polymerase to terminate {Samadashwily et al., EMBO J . 13 (1993) 4975-4983} . Here, we show that triplex-forming oligodeoxyribonucleotides (oligos) efficiently trap DNA polymerases at target DNA sequences within single-stranded (ss) templates . This was observed for all studied DNA polymerases, including Sequenase and the thermophilic Taq and Vent polymerases . The termination rate depends on the fine structure of a triplex, as well as on ambient conditions such as temperature and the concentration of magnesium ions . Inhibition of DNA synthesis was observed not only when triplexes blocked the path of DNA polymerase, but also when a polymerization primer was involved in triplex formation . Escherichia coli ss-binding (SSB) protein helps DNA polymerase overcome the triplex barrier, but with an efficiency dramatically dependent on the triplex configuration . These results describe a novel method for blocking DNA replication at target homopurine-homopyrimidine sequences by means of triplex-forming oligos in direct analogy with similar results during transcription. Nature, 1994 Nov 3, 372(6501), 111 - 3 Model for an RNA tertiary interaction from the structure of an intermolecular complex between a GAAA tetraloop and an RNA helix; Pley HW et al.; In large structured RNAs, RNA hairpins in which the strands of the duplex stem are connected by a tetraloop of the consensus sequence 5'-GNRA (where N is any nucleotide, and R is either G or A) are unusually frequent . In group I introns there is a covariation in sequence between nucleotides in the third and fourth positions of the loop with specific distant base pairs in putative RNA duplex stems: GNAA loops correlate with successive 5'-C-C.G-C base pairs in stems, whereas GNGA loops correlate with 5'-C-U.G-A . This has led to the suggestion that GNRA tetraloops may be involved in specific long-range tertiary interactions, with each A in position 3 or 4 of the loop interacting with a C-G base pair in the duplex, and G in position 3 interacting with a U-A base pair . This idea is supported experimentally for the GAAA loop of the P5b extension of the group I intron of Tetrahymena thermophila and the L9 GUGA terminal loop of the td intron of bacteriophage T4 (ref . 4) . NMR has revealed the overall structure of the tetraloop for 12-nucleotide hairpins with GCAA and GAAA loops and models have been proposed for the interaction of GNRA tetraloops with base pairs in the minor groove of A-form RNA . Here we describe the crystal structure of an intermolecular complex between a GAAA tetraloop and an RNA helix . The interactions we observe correlate with the specificity of GNRA tetraloops inferred from phylogenetic studies, suggesting that this complex is a legitimate model for intramolecular tertiary interactions mediated by GNRA tetraloops in large structured RNAs. Appl Environ Microbiol, 1994 Nov, 60(11), 3981 - 8 Cloning and sequencing of a serine proteinase gene from a thermophilic Bacillus species and its expression in Escherichia coli; Maciver B et al.; The gene for a serine proteinase from a thermophilic Bacillus species was identified by PCR amplification, and the complete gene was cloned after identification and isolation of suitably sized restriction fragments from Southern blots by using the PCR product as a probe . Two additional, distinct PCR products, which were shown to have been derived from other serine proteinase genes present in the thermophilic Bacillus species, were also obtained . Sequence analysis showed an open reading frame of 1,206 bp, coding for a polypeptide of 401 amino acids . The polypeptide was determined to be an extracellular serine proteinase with a signal sequence and prosequence . The mature proteinase possessed homology to the subtilisin-like serine proteinases from a number of Bacillus species and had 61% homology to thermitase, a serine proteinase from Thermoactinomyces vulgaris . The gene was expressed in Escherichia coli in the expression vector pJLA602 and as a fusion with the alpha-peptide of the lacZ gene in the cloning vector pGEM5 . A recombinant proteinase from the lacZ fusion plasmid was used to determine some characteristics of the enzyme, which showed a pH optimum of 8.5, a temperature optimum of 75 degrees C, and thermostabilities ranging from a half-life of 12.2 min at 90 degrees C to a half-life of 40.3 h at 75 degrees C . The enzyme was bound to a bacitracin column, and this method provided a simple, one-step method for producing the proteinase, purified to near homogeneity. Am Ind Hyg Assoc J, 1994 Nov, 55(11), 1072 - 9 Bioaerosol sampling in field studies: can samples be express mailed? Thorne PS, Lange JL, Bloebaum P, Kullman GJ. Bioaerosol sampling for viable microorganisms was conducted in 25 dairy barns in summer and in winter to examine the relationship of sample storage and shipping in determining bioaerosol concentrations separately for yeasts, molds, mesophilic bacteria, and thermophilic organisms . The study also compared the performance of three sampling methods--(1) all-glass impinger (AGI) used with peptone solution in both seasons and (2) betaine solution in winter; and (3) the nuclepore filtration and elution (NFE) method, using air filtration with subsequent elution and culturing--which were studied in a pairwise fashion with duplicate, simultaneous, side-by-side sampling . For each sample, one duplicate was analyzed within two hours in a laboratory less than 50 km from the sampling site, while the other was express-mailed to the authors' laboratory . Concentrations of all microorganisms measured by the AGI peptone method were unaffected by mailing in winter, but mesophilic bacteria increased in summer . AGI betaine samples were unchanged except for increased concentrations of molds after mailing in winter . Yeasts and mesophilic bacteria significantly decreased after mailing of NFE samples . Pairwise comparison of the sampling methods in winter yielded no significant differences in airborne concentrations for the yeasts, mesophilic bacteria, and thermophilic bacteria . Both AGI betaine and NFE methods had significantly greater concentrations of molds than AGI peptone . In summer, concentrations of yeasts and mesophilic bacteria were significantly greater with AGI peptone, as were molds with the NFE method.(ABSTRACT TRUNCATED AT 250 WORDS) FEMS Microbiol Lett, 1994 Nov 1, 123(3), 275 - 9 Cloning and sequencing of the pheU gene for tRNA(Phe) of Thermus thermophilus HB8, and genomic mapping of the pheU and pheST genes; Keller B et al.; The gene pheU for tRNA(Phe) from the extreme thermophile Thermus thermophilus HB8 was cloned and sequenced . It differed from the published tRNA(Phe) sequence by one nucleotide . Both the pheU gene and the pheST genes encoding the alpha and beta subunits of phenylalanyl-tRNA synthetase were located on the physical map of the T . thermophilus chromosome, where pheU was present on a hitherto unrecognized HpaI DNA fragment. J Bacteriol, 1994 Nov, 176(22), 6974 - 9 Characterization of a CO: heterodisulfide oxidoreductase system from acetate-grown Methanosarcina thermophila; Peer CW et al.; During the methanogenic fermentation of acetate by Methanosarcina thermophila, the CO dehydrogenase complex cleaves acetyl coenzyme A and oxidizes the carbonyl group (or CO) to CO2, followed by electron transfer to coenzyme M (CoM)-S-S-coenzyme B (CoB) and reduction of this heterodisulfide to HS-CoM and HS-CoB (A . P . Clements, R . H . White, and J . G . Ferry, Arch . Microbiol . 159:296-300, 1993) . The majority of heterodisulfide reductase activity was present in the soluble protein fraction after French pressure cell lysis . A CO:CoM-S-S-CoB oxidoreductase system from acetate-grown cells was reconstituted with purified CO dehydrogenase enzyme complex, ferredoxin, membranes, and partially purified heterodisulfide reductase . Coenzyme F420 (F420) was not required, and CO:F420 oxidoreductase activity was not detected in cell extracts . The membranes contained cytochrome b that was reduced with CO and oxidized with CoM-S-S-CoB . The results suggest that a novel CoM-S-S-CoB reducing system operates during acetate conversion to CH4 and CO2 . In this system, ferredoxin transfers electrons from the CO dehydrogenase complex to membrane-bound electron carriers, including cytochrome b, that are required for electron transfer to the heterodisulfide reductase . The cytochrome b was purified from solubilized membrane proteins in a complex with six other polypeptides . The cytochrome was not reduced when the complex was incubated with H2 or CO, and H2 uptake hydrogenase activity was not detected; however, the addition of CO dehydrogenase enzyme complex and ferredoxin enabled the CO-dependent reduction of cytochrome b. EMBO J, 1994 Nov 1, 13(21), 5113 - 20 Pulvomycin-resistant mutants of E.coli elongation factor Tu; Zeef LA et al.; This paper reports the generation of Escherichia coli mutants resistant to pulvomycin . Together with targeted mutagenesis of the tufA gene, conditions were found to overcome membrane impermeability, thereby allowing the selection of three mutants harbouring elongation factor (EF)-Tu Arg230-->Cys, Arg333-->Cys or Thr334-->Ala which confer pulvomycin resistance . These mutations are clustered in the three-domain junction interface of the crystal structure of the GTP form of Thermus thermophilus EF-Tu . This result shares similarities with kirromycin resistance; kirromycin-resistant mutations cluster in the domain 1-3 interface . Since both interface regions are involved in the EF-Tu switch mechanism, we propose that pulvomycin and kirromycin both act by specifically disturbing the allosteric changes required for the switch from EF-Tu-GTP to EF-Tu-GDP . The three-domain junction changes dramatically in the switch to EF-Tu.GDP; in EF-Tu.GDP this region forms an open hole . Structural analysis of the mutation positions in EF-Tu.GTP indicated that the two most highly resistant mutants, R230C and R333C, are part of an electrostatic network involving numerous residues . All three mutations appear to destabilize the EF-Tu.GTP conformation . Genetic and protein characterizations show that sensitivity to pulvomycin is dominant over resistance . This appears to contradict the currently accepted model of protein synthesis inhibition by pulvomycin. Biochim Biophys Acta, 1994 Nov 1, 1188(1-2), 108 - 16 Catalytic cooperativity of beef heart mitochondrial F1-ATPase revealed by using 2',3'-O-(2,4,6-trinitrophenyl)-ATP as a substrate; an indication of mutually activating catalytic sites; Muneyuki E et al.; The interaction of 2',3'-O-(2,4,6-trinitrophenyl)ATP (TNP-ATP) with bovine mitochondrial F1-ATPase (MF1) was examined under substoichiometric and stoichiometric conditions to investigate the relationship between the amount of bound TNP-AT(D)P and extent of inhibition on steady state ATP hydrolysis . The hydrolysis of bound TNP-ATP under substoichiometric condition proceeded slowly, with a first order rate constant of 0.014 s-1 . However, hydrolysis was greatly accelerated by addition of excess ATP . The hydrolyzed product, TNP-ADP, did not dissociate from the enzyme even after the addition of excess ATP . These properties were the same for both native and nucleotide depleted enzyme . The difference spectrum induced by binding TNP-ATP to MF1 had a distinct peak at 410 nm and a deep trough at 395 nm, which were similar to those induced when TNP-ATP bound to the isolated beta subunit of the thermophilic F1-ATPase . The magnitude of difference spectra as a function of TNP-ATP concentration suggested the presence of at least two types of binding sites on the MF1 molecule . The first site, where substoichiometric TNP-ATP was hydrolyzed, had a very high affinity for TNP-ATP . TNP-AT(D)P bound to this site did not dissociate even in the presence of excess ATP . TNP-AT(D)P bound to the second site dissociated slowly when excess ATP was added . The steady state ATPase activity at 100 microM ATP was linearly suppressed as pre-loaded TNP-ATP increased . The binding of 2 mol of TNP-ATP per mol of MF1 was required to abolish ATPase activity . A model which assumes mutually-activating two catalytic sites is presented to explain these results. Biokhimiia, 1994 Nov, 59(11), 1730 - 8 {A new site-specific endonuclease and methylase from a thermophilic strain of Bacillus species KT6}; Shapovalova NI et al.; The site-specific endonuclease R.BspKT6I and the cognate site-specific methylase M.BspKT6I have been isolated from the thermophilic strain of Bacillus species KT6 using gel-filtration on Sephadex G100 followed by chromatography on heparin-Sepharose and hydroxyapatite . Endonuclease BspKT6I is an isomer but not an isoschizomer of Sau3AI and MboI . It recognized on the DNA molecule the GAT decreases C sequence and cleaves it; however, unlike Sau3AI and MboI it produces 3'-protruding dinucleotides . The site cleavage is inhibited by dam-methylation . The sticky ends resulting from the BspKT6I cleavage are identical and complementary to the ends formed after the PvuI cleavage . The isolated from the B . species KT6 methylase protects the DNA from subsequent cleavage by BspKT6I . Adenine is a methylated base. Biokhimiia, 1994 Nov, 59(11), 1714 - 29 {A new site specific endonuclease-methylase from a thermophilic strain of Bacillus species LU11}; Chernov AV et al.; A new site-specific endonuclease BspLU11III was purified to homogeneity from a thermophilic strain Bacillus species LU11 . BspLU11III recognizes the 5'-GGGAC-3' sequence on the double-stranded DNA and cleaves the 10/14 and 11/15 nucleotides in different strands away from the recognition site . The enzyme exists in solution as a monomer with a molecular mass of about 93 kDa . When incubated with S-adenosyl-L-methionine, BspLU11III displays a DNA-methyltransferase activity . The adenine residue is methylated inside the recognition site 5'-GGGAC-3' in the only strand . The restriction activity does not change in the presence of ATP but is stimulated by 80 microM S-adenosyl-L-methionine (4-fold) . Magnesium cations are needed for the restriction activity . Sodium chloride stimulates the "star" activity of BspLU11III . According to its properties, BspLU11III can be classified as a type IV endonuclease. J Eukaryot Microbiol, 1994 Nov-Dec, 41(6), 546 - 53 Cilia-mediated oriented chemokinesis in Tetrahymena thermophila; Leick V et al.; The role of the cilia in the locomotion ("gliding") of Tetrahymena thermophila in a semi-solid medium has been studied when cells were migrating in gradients of attractant . Video recordings and computer-aided motion analysis of migrating cells and their ciliary activity show that Tetrahymena thermophila migrate by swimming forward in semi-solid methyl cellulose, using their cilia . Ciliary reversals occur at certain intervals and cause a termination ("stop") of cellular migration . Cells with reversed cilia resume forward migration when normal ciliary beating resumes . In gradients of attractants, cells migrating towards the attractant suppress ciliary reversals, which leads to longer runs between stops than in control cells . Cells migrating away from the attractant have a higher frequency of ciliary reversals than the control cells resulting in shorter runs . Stimulated cells adapt to a particular ambient concentration of attractant several times during migration in the gradient . Adaptation is followed by de-adaptation, which occurs during the "stop." In the presence of cycloheximide, a strong inhibitor of chemoattraction, the attractant-induced suppression of ciliary reversal is abolished (cells become desensitized to the attractant) . It is concluded that Tetrahymena has a short-term memory during adaptation . This is important for the efficiency of migration towards an attractant. J Dairy Sci, 1994 Nov, 77(11), 3287 - 95 Antimutagenicity of fermented milk; Nadathur SR et al.; Reconstituted nonfat dry milk was fermented by Lactobacillus helveticus CH65, Lactobacillus acidophilus BG2FO4, Streptococcus salivarius ssp . thermophilus CH3, Lactobacillus delbrueckii ssp . bulgaricus 191R, and by a mixture of the latter two organisms . The fermented milks were then freeze-dried, extracted in acetone, dissolved in dimethylsulfoxide, and assayed for antimutagenicity in the Ames test (Salmonella typhimurium TA 100) against N-methyl, N'-nitro, N-nitroso-guanidine, and 3,2'-dimethyl-4-amino-biphenyl . Dose-dependent activity was significant against both mutagens in all extracts . Maximal inhibitory activity against 3,2'-dimethyl-4-aminobiphenyl and N-methyl, N'-nitro, N-nitroso-guanidine was 2- and 2.7-fold greater, respectively, than that exhibited by extracts of unfermented milk . Extracts of milk fermented by L . delbrueckii ssp . bulgaricus 191R were examined further . Compounds that were responsible for activity against both mutagens were less soluble in aqueous solutions than in dimethylsulfoxide . Adjustment of milk fermented by L . delbrueckii ssp . bulgaricus 191R to pH 3, 7.6, or 13 prior to freeze-drying and acetone extraction did not significantly alter the activity specific for 3,2'-dimethyl-4-aminobiphenyl . In contrast, compounds with activity specific for N-methyl, N'-nitro, N-nitrosoguanidine were less extractable at pH 7.6 . The weak antimutagenicity of unfermented milk was not increased by addition of 2% L-lactic acid. J Dairy Sci, 1994 Nov, 77(11), 3275 - 86 Absence of cholic acid 7 alpha-dehydroxylase activity in the strains of Lactobacillus and Bifidobacterium; Takahashi T et al.; To investigate the presence of 7 alpha-dehydroxylase activity on bile acids in the bacterial strains of fermented milk products, 46 strains of Lactobacillus casei, Lactobacillus paracasei, Lactobacillus rhamnosus, Lactobacillus acidophilus, Lactobacillus gasseri, Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium breve, Bifidobacterium infantis, Bifidobacterium longum, Lactococcus lactis spp . lactis, and Streptococcus salivarius spp . thermophilus were tested for their ability to produce deoxycholic acid from cholic acid . The production of deoxycholic acid was quantitatively measured by radiochromatographic analysis in anaerobically prepared washed whole resting cells and by HPLC analysis in growing cultures . Resting whole cells from a positive control strain, Eubacterium lentum-like strain c-25, converted 81.7% of .2 mM cholic acid to deoxycholic acid and 3.7% to 7-keto-deoxycholic acid, when the cell suspension was incubated anaerobically at a concentration of 2 mg of protein/ml for 4 h at pH 7.3 . However, none of the test strains investigated in this study was able to transform cholic acid under the same conditions . In growing cultures, 91.5% of 150 micrograms/ml of cholic acid was transformed to deoxycholic acid and 1.1% to 7-keto-deoxycholic acid by E . lentum-like c-25 after a 7-d anaerobic incubation . None of the test strains showed production of either deoxycholic acid or 7-keto-deoxycholic acid as growing cultures. J Mol Evol, 1994 Nov, 39(5), 528 - 32 Using protein synthesis inhibitors to establish the phylogenetic relationships of the Sulfolobales order; Sanz JL et al.; The sensitivity of the cell-free protein synthesis systems from Acidanus brierleyi, Acidianus infernus, and Metallosphaera sedula, members of the archaeal order Sulfolobales, to 40 antibiotics with different specificities has been studied . The sensitivity patterns were compared to those of Sulfolobus solfataricus and other archaeal, bacterial, and eukaryotic systems . The comparative analysis shows that ribosomes from the sulfolobales are the most refractory to inhibitors of protein synthesis described so far . The sensitivity results have been used to ascertain in phylogenetic relationships among the members of the order Sulfolobales . The evolutionary significance of these results are analyzed in the context of the phylogenetic position of this group of extreme thermophilic microorganisms. Res Microbiol, 1994 Nov-Dec, 145(9), 651 - 8 Genotyping of Streptococcus thermophilus evidenced by restriction analysis of ribosomal DNA; Salzano G et al.; Twenty strains of Streptococcus thermophilus were classified into five different types characterized by ribotyping after DNA digestion with HindIII and hybridization with cDNA from 16S-23S rDNA of Escherichia coli . Ribotyping after digestion with HaeIII and XhoI enabled the same strains to be gathered into three groups as a consequence of the reduced influence of the flanking sequences within the analysis of rRNA operons . Amplified rDNA restriction analysis clearly confirmed the discrimination of these three different ribotypes, producing evidence to suggest that subtaxa are to be recognized within this species. Zentralbl Bakteriol, 1994 Nov, 281(4), 471 - 4 Increase of ciprofloxacin resistance in Campylobacter species in Styria, Austria; Feierl G et al.; Salmonella spp . and thermophilic Campylobacter spp . are the most important diarrhea-causing pathogens in the area investigated in Styria, Austria . The isolation rate of Campylobacter in the more than 62,000 stool specimens investigated in the six-year period between 1988 and 1993 ranged between 1.90% in 1988 and 3.58% in 1991 . The testing of susceptibility to nalidixic acid has been an usual characteristic for species identification . Nalidixic acid-resistant strains were rare in 1988-1990, but in the summer of 1991, we found an increasing number of these isolates . At the same time, we learnt about the increasing use of enrofloxacin in veterinary medicine, especially in the poultry industry, and therefore we started routine testing of Campylobacter spp . susceptibility to ciprofloxacin in 1992 . In 1992, the resistance rate to ciprofloxacin was already 16.9%, rising to 22.1% in 1993. Biol Pharm Bull, 1994 Nov, 17(11), 1446 - 50 Studies on thermophile products . X . Further biological properties of isofatty acid-containing phosphatidylglycerol that enhances the induction of suppressor T cells; Kohama Y et al.; Isofatty acid-containing phosphatidylglycerol (Fr . 7-C), isolated from Bacillus stearothermophilus UBT8038, enhances the induction of concanavalin A (Con A)-activated suppressor T (Ts) cells in a dose dependent manner (0.01-1 microgram/ml) . Its further biological properties on mouse mixed lymphocyte reaction (MLR) has been demonstrated . Fr . 7-C (0.01-1 microgram/ml) suppressed the MLR at 4 d in a dose-dependent manner when added at the start of splenocyte cultivation . Moreover, Fr . 7-C was effective in preventing the generation of cytotoxic T lymphocytes after the MLR . On the other hand, this fraction significantly enhanced the induction of Ts cells in the MLR carried out in any of the antigen-specific, antigen-nonspecific and major histocompatibility complex antigen-nonrestricted fashions . Fr . 7-C increased prostaglandin E2 (PGE2) release approximately 2-fold in the culture supernatant of Con A-activated splenocytes, and PGE2 release decreased dose-dependently when cultured with indomethacin . The inhibitory effect by Fr . 7-C on the MLR was abrogated by the addition of indomethacin . The enhancement by Fr . 7-C on Ts cell induction was blocked by indomethacin in a dose dependent manner . These results strongly suggest that Fr . 7-C suppresses the MLR via the enhancement of antigen-nonspecific Ts cell induction mediated at least partly by PGE2. Protein Sci, 1994 Nov, 3(11), 2097 - 103 Probing protein-cofactor interactions in the terminal oxidases by second derivative spectroscopy: study of bacterial enzymes with cofactor substitutions and heme A model compounds; Felsch JS et al.; Second derivative absorption spectra are reported for the aa3-cytochrome c oxidase from bovine cardiac mitochondria, the aa3-600 ubiquinol oxidase from Bacillus subtilis, the ba3-cytochrome c oxidase from Thermus thermophilis, and the aco-cytochrome c oxidase from Bacillus YN-2000 . Together these enzymes provide a range of cofactor combinations that allow us to unequivocally identify the origin of the 450-nm absorption band of the terminal oxidases as the 6-coordinate low-spin heme, cytochrome a . The spectrum of the aco-cytochrome c oxidase further establishes that the split Soret band of cytochrome a, with features at 443 and 450 nm, is common to all forms of the enzyme containing ferrocytochrome a and does not depend on ligand occupancy at the other heme cofactor as previously suggested . To test the universality of this Soret band splitting for 6-coordinate low-spin heme A systems, we have reconstituted purified heme A with the apo forms of the heme binding proteins, hemopexin, histidine-proline-rich glycoprotein and the H64V/V68H double mutant of human myoglobin . All 3 proteins bound the heme A as a (bis)histidine complex, as judged by optical and resonance Raman spectroscopy . In the ferroheme A forms, none of these proteins displayed evidence of Soret band splitting . Heme A-(bis)imidazole in aqueous detergent solution likewise failed to display Soret band splitting . When the cyanide-inhibited mixed-valence form of the bovine enzyme was partially denatured by chemical or thermal means, the split Soret transition of cytochrome a collapsed into a single band at 443 nm.(ABSTRACT TRUNCATED AT 250 WORDS) J Biol Chem, 1994 Oct 28, 269(43), 27074 - 9 Isolation of the stable hexameric DnaK.DnaJ complex from Thermus thermophilus; Motohashi K et al.; A DnaK homolog (T.DnaK) has been purified as a stable complex with a DnaJ homolog (T.DnaJ) from a thermophilic bacterium, Thermus thermophilus . This complex has an approximate molecular size of 300 kDa and appears to contain three copies of each of T.DnaK and T.DnaJ molecules . Consistently, trigonal ring structures with a diameter (trigonal apex-to-apex) of about 11 nm were observed with electron microscopy . The complex has no endogenously bound AT(D)P and is stable in the presence of Mg-AT(D)P . It possesses a weak ATPase activity and retains about 3 mol of ADP/mole of the complex when incubated with Mg-ATP . This complex is able to interact with the reduced carboxymethylated alpha-lactalbumin which we used as a model unfolded protein. Nucleic Acids Res, 1994 Oct 25, 22(21), 4432 - 40 Identification of DNA-binding proteins that recognize a conserved type I repeat sequence in the replication origin region of Tetrahymena rDNA; Umthun AR et al.; An origin of DNA replication has been mapped within the 5' non-transcribed spacer region of the amplified macronuclear rRNA genes (rDNA) of Tetrahymena thermophila . Mutations in 33 nt conserved AT-rich Type I repeat sequences located in the origin region cause defects in the replication and/or maintenance of amplified rDNA in vivo . Fe(II)EDTA cleavage footprinting of restriction fragments containing the Type I repeat showed that most of the conserved nucleotides were protected by proteins in extracts of Tetrahymena cells . Two classes of proteins that bound the Type I repeat were identified and characterized using synthetic oligonucleotides in electrophoretic mobility shift assays . One of these, ds-TIBF, bound preferentially to duplex DNA and exhibited only moderate specificity for Type I repeat sequences . In contrast, a single-stranded DNA-binding protein, ssA-TIBF, specifically recognized the A-rich strand of the Type I repeat sequence . Deletion of the 5' or 3' borders of the conserved sequence significantly reduced binding of ssA-TIBF . The binding properties of ssA-TIBF, coupled with genetic evidence that Type I sequences function as cis-acting rDNA replication control elements in vivo, suggest a possible role for ssA-TIBF in rDNA replication in Tetrahymena. Biochim Biophys Acta, 1994 Oct 19, 1208(2), 310 - 5 Indole-3-glycerol-phosphate synthase from Sulfolobus solfataricus as a model for studying thermostable TIM-barrel enzymes; Andreotti G et al.; Indole-3-glycerol-phosphate synthase, a thermophilic and thermostable enzyme from the archaeon Sulfolobus solfataricus, was purified and characterized . The sequence of the thermophilic enzyme was compared to the sequence of a homologous mesophilic enzyme from Escherichia coli . The secondary structure of the thermophilic enzyme was predicted taking into account the patterns of hydropathy, chain flexibility and amphipathicity and the CD spectrum . From this analysis it turned out that indole-3-glycerol-phosphate synthase from S . solfataricus can be considered a model for studying thermostable TIM-barrel enzymes . Some peculiarities of the amino-acid sequence of indole-3-glycerol-phosphate synthase from S . solfataricus are discussed in relation to the thermostability of the enzyme. Biochim Biophys Acta, 1994 Oct 18, 1219(2), 559 - 62 Nucleotide sequence of the phosphotransacetylase gene of Escherichia coli strain K12; Matsuyama A et al.; The phosphotransacetylase gene (pta) from Escherichia coli strain K-12 1100 was identified in a cloned fragment of chromosomal DNA (Yamamoto-Otake, H., Matsuyama, A . and Nakano, A . (1990) Appl . Microbiol . Biotechnol . 33, 680-682) . Overexpression in E . coli confirmed the presence of the pta gene within the cloned fragment . DNA sequence analysis of the cloned pta gene indicates that the predicted phosphotransacetylase polypeptide chain is 713 amino acids in length . The carboxyterminal region of the E . coli phosphotransacetylase shows 42.6% sequence identity with the corresponding enzyme from Methanosarcina thermophila (142 out of 333 residues in corresponding positions are identical) . Several short regions of high sequence identity may be structurally or functionally important for enzymic activity. EMBO J, 1994 Oct 17, 13(20), 4863 - 9 Contribution of structural elements to Thermus thermophilus ribonuclease P RNA function; Schlegl J et al.; We have performed a deletion and mutational analysis of the catalytic ribonuclease (RNase) P RNA subunit from the extreme thermophilic eubacterium Thermus thermophilus HB8 . Catalytic activity was reduced 600-fold when the terminal helix, connecting the 5' and 3' ends of the molecule, was destroyed by deleting 15 nucleotides from the 3' end . In comparison, the removal of a large portion (94 nucleotides, about one quarter of the RNA) of the upper loop region impaired function only to a relatively moderate extent (400-fold reduction in activity) . The terminal helix appears to be crucial for the proper folding of RNase P RNA, possibly by orientating the adjacent universally conserved pseudoknot structure . The region containing the lower half of the pseudoknot structure was shown to be a key element for enzyme function, as was the region of nucleotides 328-335 . Deleting a conserved hairpin (nucleotides 304-327) adjacent to this region and replacing the hairpin by a tetranucleotide sequence or a single cytidine reduced catalytic activity only 6-fold, whereas a simultaneous mutation of the five highly conserved nucleotides in the region of nucleotides 328-335 reduced catalytic activity by > 10(5)-fold . The two strictly conserved adenines 244 and 245 (nucleotides 248/249 in Escherichia coli RNase P RNA) were not as essential for enzyme function as suggested by previous data . However, additional disruption of two helical segments (nucleotides 235-242) adjacent to nucleotides 244 and 245 reduced activity by > 10(4)-fold, supporting the notion that nucleotides in this region are also part of the active core structure.(ABSTRACT TRUNCATED AT 250 WORDS) FEMS Microbiol Lett, 1994 Oct 15, 123(1-2), 55 - 61 Molecular cloning and sequence analysis of the proBA operon from an extremely thermophilic eubacterium Thermus thermophilus; Kosuge T et al.; A 3.6 kb DNA fragment carrying the Thermus thermophilus proBA region, which encodes the first two steps in the proline biosynthetic pathway, was cloned from the Thermus thermophilus gene library, and its complete nucleotide sequence was determined . The deduced amino acid sequence of gamma-glutamyl kinase (40,657 Da), the product of proB gene, and gamma-glutamyl phosphate reductase (48,747 Da), the product of proA gene, showed 44.1% and 44.4% identity to those of Escherichia coli, respectively . The termination codon of the proB gene and the initiation codon of the proA gene overlapped by 2 bp . A possible transcriptional termination structure was found downstream of the proA gene but not downstream of the proB gene . These results indicate that the proBA genes of T . thermophilus form a single operon as in E . coli. J Biol Chem, 1994 Oct 14, 269(41), 25928 - 35 Cloning, sequencing, and expression of RecA proteins from three distantly related thermophilic eubacteria; Wetmur JG et al.; Sequences of the recA genes of the highly divergent thermopholic eubacteria Thermus aquaticus (and Thermus thermophilus), Thermotoga maritima, and Aquifex pyrophilus were determined from fragments derived by polymerase chain reaction (PCR) with degenerate primers and from inverse PCR products obtained using unique primers based on the fragment sequences . The source of the PCR products was verified by Southern hybridization . Complete PCR-derived recA genes were cloned into an expression vector regulated by a temperature-sensitive lambda-repressor, and independently derived clones expressing thermostable recA were selected . DNA sequences were verified to be authentic by direct cycle-sequencing of PCR products and/or sequencing of several clones . In contrast to Escherichia coli RecA protein, all the purified thermophilic RecA proteins exhibited single-stranded DNA-dependent ATPase activity optima above 70 degrees C . Phylogenetic analysis of RecA sequences suggested that the thermophilic RecA proteins were at least as different from one another as were Gram-positive organisms, mesophilic Gram-negative organisms, and cyanobacteria . In spite of substantial sequence divergence, interesting characteristics of the thermostable RecA proteins included increased valine content, common amino acid replacements at two highly conserved sites, and an increase in the calculated isoelectric point of approximately a full pH unit. J Chromatogr B Biomed Appl, 1994 Oct 14, 660(2), 223 - 33 Detection of ribose-methylated nucleotides in Pyrodictium occultum tRNA by liquid chromatography--frit-fast atom bombardment mass spectrometry; Takeda N et al.; Ribose-methylated dinucleotides of the type NmpN' derived from digestion of tRNA with RNase T2 were separated and characterized by directly combined liquid chromatography--mass spectrometry (LC-MS) with a continuous-flow frit-fast atom bombardment (frit-FAB) interface . Prediction of NmpN' peaks was readily made by comparison of the LC profile with that of comparative nuclease P1 digest . The identity of the candidate peaks including NmpN' was further recognized by the mass spectra, in which NmpN' showed intense molecular-related ions, in addition to sequence-specific fragment ions, to verify the chemical structures in both positive- and negative-ion modes . The method was applied to screening NmpN' (and NmpN' mpN") in tRNA from the extremely thermophilic archaeon Pyrodictium occultum. Nature, 1994 Oct 13, 371(6498), 619 - 22 Ribozyme-mediated repair of defective mRNA by targeted, trans-splicing; Sullenger BA et al.; Ribozymes can be targeted to cleave specific RNAs, which has led to much interest in their potential as gene inhibitors . Such trans-cleaving ribozymes join a growing list of agents that stop the flow of genetic information . Here we describe a different application of ribozymes for which they may be uniquely suited . By targeted trans-splicing, a ribozyme can replace a defective portion of RNA with a functional sequence . The self-splicing intron from Tetrahymena thermophila was previously shown to mediate trans-splicing of oligonucleotides in vitro . As a model system for messenger RNA repair, this group I intron was re-engineered to regenerate the proper coding capacity of short, truncated lacZ transcripts . Trans-splicing was efficient in vitro and proceeded in Escherichia coli to generate translatable lacZ messages . Targeted trans-splicing represents a general means of altering the sequence of specified transcripts and may provide a new approach to the treatment of many genetic diseases. J Mol Biol, 1994 Oct 7, 242(5), 709 - 11 Crystallization of mutant beta subunit of F1-ATPase from thermophilic Bacillus PS3; Saika K et al.; The mutant beta subunit of F1-ATPase from a thermophilic Bacillus strain, PS3, in which tyrosine at position 341 is replaced by leucine (beta Y341L) was expressed in Escherichia coli and crystallized by the vapor-diffusion procedure . Small needle-like crystals were obtained using ammonium sulfate as a precipitant and grown by the stepwise seeding method . The crystals obtained by this procedure diffracted X-rays to about 3 A resolution . The diffraction patterns indicated that the crystals belong to the orthorhombic system and the space group I222 or I2(1)2(1)2(1) with unit-cell dimensions of a = 232 A, b = 66 A, and c = 80 A . It is thought that the asymmetric unit comprises one beta Y341L molecule. J Biol Chem, 1994 Oct 7, 269(40), 24762 - 9 Purification and characterization of extremely thermophilic and thermostable 5'-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus . Purine nucleoside phosphorylase activity and evidence for intersubunit disulfide bonds; Cacciapuoti G et al.; 5'-Methylthioadenosine phosphorylase from Sulfolobus solfataricus, a thermoacidophilic archaeon optimally growing at 87 degrees C, has been purified to homogeneity . Reducing agents are not required for catalytic activity . The enzyme has a molecular mass of 160 kDa and is composed of six apparently identical subunits of 27 kDa . The NH2-terminal sequence shows high homology (50%) with the NH2-terminal sequence of Escherichia coli purine nucleoside phosphorylase . Physicochemical and kinetic features are reported . 5'-Methylthioadenosine phosphorylase is highly thermophilic, with an optimum temperature of 120 degrees C . The enzyme is characterized by extreme thermal stability, remaining completely active after 2 h at 100 degrees C and showing half-inactivation times of 15 and 5 min when incubated at 130 and 140 degrees C, respectively . An apparent melting temperature of 132 degrees C has been calculated . After 24 h of incubation at room temperature no loss of activity is detected in the presence of 9 M urea, 4 M guanidine hydrochloride, 0.075% SDS, 50% methanol, 50% ethanol, 50% dimethylformamide, 1 M NaCl, and 1% Triton X-100 . Data are also reported on the enzyme's resistance to proteolysis and on the effect of salts, detergents, solvents, and reducing agents on enzyme thermostability . Labeling experiments with iodo{2-14C}acetic acid resulted in the incorporation of approximately 12 mol of labeled iodoacetate/mol of protein, indicating the presence of six disulfide bonds that, on the basis of SDS-polyacrylamide gel electrophoresis, are probably positioned intersubunits, resulting in the organization of the enzyme into two trimers . 5'-Methylthioadenosine (MTA) phosphorylase is endowed with a broad substrate specificity, being able to phosphorolytically cleave inosine, guanosine, and adenosine with a better efficiency than MTA, allowing us to hypothesize that in S . solfataricus the same enzyme is responsible for the catabolism of MTA and of these purine nucleosides. Appl Environ Microbiol, 1994 Oct, 60(10), 3764 - 73 Cloning of the aapT gene and characterization of its product, alpha-amylase-pullulanase (AapT), from thermophilic and alkaliphilic Bacillus sp . strain XAL601; Lee SP et al.; A thermophilic and alkaliphilic Bacillus sp . strain, XAL601, was isolated from soil . It produces a thermostable and alkaline-stable enzyme with both alpha-amylase and pullulanase activities . The alpha-amylase-pullulanase gene (aapT) from this Bacillus strain was cloned, and its nucleotide sequence was determined (GenBank accession number D28467) . A very large open reading frame composed of 6,096 bases, which encodes 2,032 amino acid residues with an M(r) of 224,992, was found . The deduced amino acid sequence revealed that the four highly conserved regions that are common among amylolytic enzymes were well conserved . These include an active center and common substrate-binding sites of various amylases . In the C-terminal region, a six-amino-acid sequence (Gly-Ser-Gly-Thr-Thr-Pro) is repeated 12 times . The aapT gene was then subcloned in Escherichia coli and overexpressed under the control of the lac promoter . Purification of AapT from this recombinant E . coli was performed, and it was shown that the aapT gene product exhibits both alpha-amylase and pullulanase activities with one active site . The optimum temperature and pH for enzyme activity were found to be 70 degrees C and pH 9, respectively . Furthermore, AapT was found to strongly adsorb to crystalline cellulose (Avicel) and raw corn starch . Final hydrolyzed products from soluble starch range from maltose (G2) to maltotetraose (G4) . Only maltotriose (G3) was produced from pullulan . The enzyme also hydrolyzes raw starch under a broad range of conditions (60 to 70 degrees C and pH 8 to 9). Appl Environ Microbiol, 1994 Oct, 60(10), 3579 - 84 Stabilization and rational design of serine protease AprM under highly alkaline and high-temperature conditions; Masui A et al.; Rational shift of the optimum pH toward alkalinity and enhancement of thermostability were investigated by using a thermostable extremely alkaline protease (optimum pH, 12 to 13) from the alkaliphilic and thermophilic Bacillus sp . strain B18' . The protease gene (aprM) was cloned, and the sequence analysis revealed an open reading frame of 361 amino acids that was composed of a putative signal sequence (24 amino acids), a prosequence (69 amino acids), and a mature enzyme (268 amino acids) (molecular weight, 27,664) . The amino acid sequence of this protease was compared with those of other serine proteases . A direct correlation of higher optimum pH with an increase in the number of arginine residues was observed . An even more thermostable mutant enzyme was created by introducing a point mutation . When the position of the beta-turn, Thr-203, was replaced by Pro, the residual activity of this mutant enzyme at 80 degrees C for 30 min was higher than that of the wild-type enzyme (50% versus 10%) . The specific activity of this mutant enzyme at 70 degrees C was 105% of that of the wild-type enzyme under nondenaturation condition . These data suggest that the higher content of Arg residues favors the alkalinity of the serine protease and that introduction of a Pro residue into the beta-turn structure stabilizes the enzyme. Int J Syst Bacteriol, 1994 Oct, 44(4), 620 - 6 Phylogenetic position of the genus Hydrogenobacter; Pitulle C et al.; The genus Hydrogenobacter consists of extremely thermophilic, obligately chemolithotrophic organisms that exhibit anaerobic anabolism but aerobic catabolism . Preliminary studies of the phylogenetic position of these organisms based on limited 16S ribosomal DNA sequence data suggested that they belong to one of the earliest branching orders of the Bacteria . In this study, the complete 16S ribosomal DNA sequences of two type strains, Hydrogenobacter thermophilus TK-6 and Calderobacterium hydrogenophilum Z-829, and another isolate, Hydrogenobacter sp . strain T3, were determined, and the phylogenetic positions of these organisms were examined . Our results revealed that the two type strains are members of a single genus, the genus Hydrogenobacter . Our results also verified the previous conclusion that the Aquifex-Hydrogenobacter complex belongs to a very early branching order, the "Aquificales." Within this order, the relationships among the various organisms are such that only a single family, the "Aquificaceae," can be recognized at this time . Given the early branching point of the "Aquificales," the characteristics of these organisms support the view that the last common ancestor of existing life was thermophilic and suggest that this ancestor may have fixed carbon chemoautotrophically. Dev Biol, 1994 Oct, 165(2), 418 - 31 Identification of a novel polypeptide involved in the formation of DNA-containing vesicles during macronuclear development in Tetrahymena; Madireddi MT et al.; An abundant phosphoprotein with an apparent molecular mass of 65 kDa (p65) has been identified that is enriched in developing new macronuclei (or anlagen) isolated from the holotrichous ciliate, Tetrahymena thermophila . During early stages of macronuclear development, p65 is actively synthesized and deposited into young (4C) anlagen and is not found in micronuclei or parental macronuclei . p65 is not detected in older (8C) anlagen or in vegetatively growing or starved cells, and thus p65 is under stringent developmental control . In situ analyses, using polyclonal antibodies generated against p65, demonstrate that p65 undergoes a pronounced change in distribution during anlagen differentiation . Initially, anlagen are uniformly stained with these antibodies . However, following separation of conjugants, this staining pattern converts to one that is punctate and fragmented . As development proceeds, most, if not all, of these p65-based particles become peripherally located in anlagen and appear as well-defined vesicles surrounding a discrete central core of DNA of yet undetermined origin . This remarkable cytological distribution suggests an involvement of p65 in the elimination or processing of DNA during anlagen differentiation . If the above is correct, p65 provides the first inroad into the protein machinery involved in the well-known DNA rearrangements that characterize ciliate macronuclear development. J Bacteriol, 1994 Oct, 176(20), 6238 - 44 Properties of a pentapeptide inhibitor of peptidyltransferase that is essential for cat gene regulation by translation attenuation; Gu Z et al.; Inducible chloramphenicol resistance genes cat and cmlA are regulated by translation attenuation . For both genes, the leader codons that must be translated to deliver a ribosome to the induction site specify a peptide that inhibits peptidyltransferase in vitro . The antipeptidyltransferase activity of the peptides is thought to select the site of ribosome stalling that is essential for induction . Using variations of the cat-86 leader-encoded 5-mer peptide MVKTD, we demonstrate a correlation between the in vitro antipeptidyltransferase activity and the ability of the same peptide to support induction by chloramphenicol in vivo . MVKTD footprints to nucleotides 2058, 2059, and 2060 in 23S rRNA . In vivo methylation of nucleotide 2058 by the ermC methylase interferes neither with cat-86 induction nor with peptide inhibition of peptidyltransferase . The methylation eliminates the competition that normally occurs in vitro between erythromycin and MVKTD . MVKTD inhibits the peptidyltransferase of several eubacteria, a representative Archaea species, and the eukaryote Saccharomyces cerevisiae . Bacillus stearothermophilus supports the in vivo induction of cat-86, and the RNA that is phenol extracted from the 50S ribosomes of this gram-positive thermophile is catalytically active in the peptidyltransferase assay and sensitive to peptide inhibition . Our results indicate that peptidyltransferase inhibition by a cat leader peptide is essential to induction, and this activity can be altered by minor changes in the amino acid sequence of the peptide . The broad range of organisms shown to possess peptide-inhibitable peptidyltransferase suggests that the target is a highly conserved component of the ribosome and includes 23S rRNA. J Bacteriol, 1994 Oct, 176(20), 6165 - 9 The sequence of the single 16S rRNA gene of the thermophilic eubacterium Rhodothermus marinus reveals a distant relationship to the group containing Flexibacter, Bacteroides, and Cytophaga species; Andresson OS et al.; Rhodothermus marinus, a gram-negative heterotrophic marine thermophile, has been the subject of several recent studies . Isolation, sequencing, and analyses of a 16S rRNA gene have shown that R . marinus diverges sharply from major bacterial phyla and is most closely allied to the Flexibacter-Cytophaga-Bacteroides group . Further analyses revealed that the R . marinus chromosome contains a single rRNA operon with a 16S-23S intergenic region coding for tRNA(Ile) and tRNA(Ala). J Bacteriol, 1994 Oct, 176(19), 6148 - 52 Acquired thermotolerance and heat shock proteins in thermophiles from the three phylogenetic domains; Trent JD et al.; Thermophilic organisms from each of the three phylogenetic domains (Bacteria, Archaea, and Eucarya) acquired thermotolerance after heat shock . Bacillus caldolyticus grown at 60 degrees C and heat shocked at 69 degrees C for 10 min showed thermotolerance at 74 degrees C, Sulfolobus shibatae grown at 70 degrees C and heat shocked at 88 degrees C for 60 min showed thermotolerance at 95 degrees C, and Thermomyces lanuginosus grown at 50 degrees C and heat shocked at 55 degrees C for 60 min showed thermotolerance at 58 degrees C . Determinations of protein synthesis during heat shock revealed differences in the dominant heat shock proteins for each species . For B . caldolyticus, a 70-kDa protein dominated while for S . shibatae, a 55-kDa protein dominated and for T . lanuginosus, 31- to 33-kDa proteins dominated . Reagents that disrupted normal protein synthesis during heat shock prevented the enhanced thermotolerance. J Dairy Sci, 1994 Oct, 77(10), 2880 - 9 Purification and characterization of a general aminopeptidase (St-PepN) from Streptococcus salivarius ssp . thermophilus CNRZ 302; Rul F et al.; A general aminopeptidase (St-PepN) was purified from an intracellular extract of Streptococcus salivarius ssp . thermophilus CNRZ 302 by ion-exchange chromatography and hydrophobic interaction chromatography . Gel electrophoresis of the purified enzyme in denaturing or nondenaturating conditions showed a single protein band . The enzyme is a monomer with a molecular mass of 97 kDa . Its activity is maximal at pH 7 and 36 degrees C and is completely abolished by CuCl2 and ZnCl2 . The enzyme is strongly inhibited by metal-chelating reagents, such as EDTA and o-phenanthroline, which suggests that St-PepN is a metalloenzyme . The enzyme showed activity toward p-nitroanilide derivatives or dipeptides and tripeptides and showed a preference for hydrophobic or basic amino acids at the N-terminal position . Longer peptide chains, such as the B-chain of insulin, glucagon, or peptides generated by the hydrolysis of caseins, were degraded, too . The sequence of the first 21 residues of the mature enzyme was determined and showed high homology with that of the aminopeptidase PepN isolated from Lactococcus lactis ssp . cremoris Wg2 . The properties of the enzyme are compared with those of corresponding enzymes of other species of lactic acid bacteria. Genetics, 1994 Oct, 138(2), 297 - 301 A temperature-sensitive mutation of the temperature-regulated SerH3 i-antigen gene of Tetrahymena thermophila: implications for regulation of mutual exclusion; LaCrosse GL et al.; The Ser genes of Tetrahymena thermophila specify alternative forms of a major cell surface glycoprotein, the immobilization or i-antigen (i-ag) . Regulation of i-ag expression assures that at least one i-ag gene is expressed at all times . To learn more about the regulatory system and the possible role of i-ag itself, we studied SerH3-ts1, a temperature-sensitive allele of the temperature-regulated SerH3 gene normally expressed from 20-36 degrees . In homozygotes grown at the nonpermissive temperature (> 32 degrees), H3 is not present on the cell surface, but the gene continues to be transcribed until its 36 degrees cutoff . H3 formed at the permissive temperature is stable at nonpermissive temperatures, indicating that SerH3-ts1 is temperature-sensitive for synthesis rather than function . At nonpermissive temperatures, the S i-ag is expressed in place of H3 . This result suggests that normal H protein may play a role in regulating S expression . SerH3-ts1 was isolated following mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) . Sequencing of SerH3-ts1 revealed a single A --> G transition at nucleotide 473, resulting in the substitution of glycine for aspartate . The affected residue is conserved in the internal repeats comprising the H protein, and the charge difference correlates with changes in electrophoretic mobility of the H3 protein. J Mol Biol, 1994 Sep 30, 242(4), 397 - 407 The molecular chaperonin TF55 from the Thermophilic archaeon Sulfolobus solfataricus . A biochemical and structural characterization; Knapp S et al.; The purification and characterization of a new type of thermostable chaperonin from the archaebacterium Sulfolobus solfataricus is described . The chaperonin forms a hetero-oligomeric complex of two different, but closely related, subunits, which we have assigned TF55-alpha and TF55-beta . Their N-terminal sequences and amino acid residue compositions are reported . Two-dimensional projections of the chaperonin have been reconstructed from electron microscopy images, showing a 9-fold symmetrical complex, about 17.5 nm in height and 16 nm in diameter, with a central cavity of 4.5 nm . The complex is resistant to denaturing agents at room temperature and only pH values lower than 2 lead to dissociation . The separated subunits do not reassemble spontaneously but require Mg2+ and ATP for complex formation . Both subunits are necessary for formation of the TF55 oligomer . Significant structural changes have been observed after phosphorylation, thus providing evidence for a structural mobility during the chaperonin-assisted folding process of a protein . The phosphorylation reaction is modulated by potassium and magnesium ions . Magnesium seems to have an inhibitory effect, whereas potassium enhances this reaction. Biochemistry, 1994 Sep 20, 33(37), 11340 - 8 Fluorescence-detected stopped flow with a pyrene labeled substrate reveals that guanosine facilitates docking of the 5' cleavage site into a high free energy binding mode in the Tetrahymena ribozyme; Bevilacqua PC et al.; Fluorescence-detected stopped flow kinetics are reported for binding of pyrene (pyr) labeled oligonucleotide substrates, pyrCUCUA and pyrCCUCUA, to the L-21 ScaI ribozyme from Tetrahymena thermophila . Both oligomer substrates contain a UA sequence that mimics the cleavage site where pG attacks the self-splicing group I intron from which the ribozyme was derived . Kinetics were measured in the presence and absence of saturating 5'-monophosphate guanosine substrate (pG) at 5 mM Mg2+ and 15 degrees C . In the absence of pG, binding of both oligonucleotide substrates is consistent with a one step mechanism involving only base pairing . Upon addition of pG, pyrCCUCUA is observed to bind in two steps: base pairing to the ribozyme to form the P1 helix and, presumably, subsequent docking of the P1 helix into the catalytic core . A third transient is also observed, which likely includes the chemical step following docking . All rate constants are measured for this mechanism . Surprisingly, the equilibrium constant for docking, K2, is unfavorable in the absence of pG (K2 < 1) and only modestly favorable in the presence of pG (K2 = 4) . These results contrast with those for a 5' exon mimic, pyrCCUCU, in which docking is strongly favored under the above conditions in the absence of pG; K2 = 100 {Bevilacqua, P . C., Kierzek, R., Johnson, K . A., & Turner, D . H . (1992) Science 258, 1355-1358} . These results suggest an unfavorable interaction between the ribozyme and the pA at the site of cleavage . Implications are discussed for the catalytic strategy of the ribozyme and for the self-splicing cascade that occurs in nature. Biochemistry, 1994 Sep 20, 33(37), 11200 - 8 Ultrafast energy transfer in FMO trimers from the green bacterium Chlorobium tepidum; Savikhin S et al.; Time-resolved absorption difference profiles were obtained for FMO trimers, isolated from the green thermophilic bacterium Chlorobium tepidum, using one- and two-color femtosecond pump-probe techniques . Uphill and downhill energy transfers between inequivalent pigments in this antenna contribute to lifetime components that range from approximately 100 to approximately 900 fs in the isotropic absorption difference signals, depending on the pump and probe wavelengths . Vibrational thermalization of BChl a pigments may also influence the kinetics . The major lifetime components in the anisotropy decays at most wavelengths are 75-135 fs and 1.4-2.0 ps . The slower anisotropy decays probably stem from equilibration among equivalent, lowest-energy pigments belonging to different subunits in the trimer . The initial anisotropy r(0) is appreciably larger than 0.4 at several wavelengths, but r(t) typically decays to a value less than 0.4 within approximately 100 fs. Gene, 1994 Sep 15, 147(1), 101 - 6 Identification and sequencing of the Thermotoga maritima lacZ gene, part of a divergently transcribed operon; Moore JB et al.; The lacZ gene encoding a beta-galactosidase (beta Gal) from the hyperthermophile Thermotoga maritima was cloned on an 11-kb fragment by complementation of an Escherichia coli lacZ deletion stain . The nucleotide sequence of the structural gene and two other ORFs found within a 6317-bp region were determined . The deduced amino acid (aa) sequence of the Tt . maritima beta Gal predicts a 1037-aa polypeptide with a calculated M(r) of 122,312 . The translated sequence is 30% similar to nine other beta Gal sequences from bacteria and one yeast . Alignment of the Tt . maritima beta Gal with these other sequences reveals that the residues responsible for Mg2+ binding, catalysis and substrate recognition are conserved in the thermophilic enzyme . Sequence analysis also revealed the presence of a divergently transcribed operon containing at least two other genes 5' to lacZ . These ORFs encode proteins homologous to a second family of beta Gal found in Bacillus species and to an ATP-dependent family of bacterial oligopeptide transport proteins. FEMS Microbiol Lett, 1994 Sep 15, 122(1-2), 85 - 90 Isolation and characterisation of promoter regions from Streptococcus thermophilus; Constable A et al.; Four promoter regions required for the expression of a promoterless antibiotic resistance gene (cat194) in Streptococcus thermophilus were isolated by random chromosomal cloning experiments . These were shown to be functional in vivo, and their sequences were determined . Each region expressed different amounts of Cat protein as determined by enzyme activities . One region, STP10, was found to contain the 5' coding region of the large ribosomal subunit protein L20. Eur J Biochem, 1994 Sep 15, 224(3), 923 - 30 Cloning and sequencing of a Rhodothermus marinus gene, bglA, coding for a thermostable beta-glucanase and its expression in Escherichia coli; Spilliaert R et al.; A gene library of the thermophilic eubacterium, Rhodothermus marinus, strain 21, was prepared in pUC18 and used to transform Escherichia coli . Of 5400 transformants, two produced halos on lichenan plates after Congo-red staining . Restriction mapping showed that the two clones shared an overlapping 1200-bp DNA fragment, which was used for DNA sequencing . Five potential methionine (Met) translational-initiation codons were identified . A putative signal peptide of 30 amino acids was identified with a hydrophobic core of nine hydrophobic amino acids . The molecular mass of the mature enzyme was estimated to be 29.7 kDa . A comparison of the primary protein sequence of beta-glucanase of Rhodothermus marinus with other glycosyl hydrolases showed 38.5% identity to the C-terminal part of the beta-1,3-glucanase of Bacillus circulans and limited identity to bacterial endo-beta-1,3-1,4-glucanases . The amino acid sequence showed high similarity to regions surrounding the catalytic Glu residue of bacterial beta-glucanases . A gene fragment of 889 bp containing the catalytic domain was overexpressed in E . coli using the pET23, T7-phage RNA polymerase system . The enzyme showed activity on lichenan, beta-glucan and laminarin but not on CMC cellulose or xylan . The expressed enzyme was purified by heat treatment of the host . The enzyme had a temperature and pH optima of 85 degrees C and pH 7.0, respectively, and was shown to retain full activity after incubation for 16 h at 80 degrees C and have a half life of 3 h at 85 degrees C. Structure, 1994 Sep 15, 2(9), 785 - 8 The ABC of EF-G; Jurnak F; The recently solved crystal structures of Thermus thermophilus elongation factor G, with and without GDP, reveal a protein of five domains with surprising features which can be correlated with biochemical data to suggest probable functional roles. Biochim Biophys Acta, 1994 Sep 13, 1219(1), 107 - 14 DNA unknotting activity (DNA topoisomerase II) isolated from a thermophilic archaebacterium Sulfolobus is inhibited by novobiocin . Partial purification, identification of the two subunits and characteristics of the enzyme; Assairi LM; DNA topoisomerases II are enzymes which have been purified from a lot of organisms and have been found to be involved in segregation of chromosomes . The following article reports the analysis of a partially purified DNA topoisomerase II from Sulfolobus (strain B12) a thermophilic archaebacterium which grows at 80 degrees C . The enzyme is composed by two subunits: the A subunit with a molecular mass of 85,000 Da which contains the nicking-closing activity and the B subunit with a molecular mass of 65,000 Da which contains the ATP binding site . The enzyme relaxes negatively as well as positively supercoiled DNA consequently to ATP hydrolysis into ADP (as eukaryotic DNA topoisomerases II and Escherichia coli DNA topoisomerase IV do) . DNA relaxation catalyzed by the thermophilic enzyme is inhibited in the presence of both bacterial antibiotics acting at the ATP binding site such as novobiocin and coumermycin A1 at the concentration which was found to inhibit the E . coli type II DNA topoisomerases (DNA gyrase and DNA topoisomerase IV) . Based on the relaxation of both negatively and positively supercoiled DNA and the sensitivity to antibiotics such as novobiocin and coumermycin A1, the DNA topoisomerase II isolated from thermophilic archaebacterium shares common characteristics with E . coli DNA topoisomerase II. FEBS Lett, 1994 Sep 12, 351(3), 385 - 8 An Escherichia coli cyoE gene homologue in thermophilic Bacillus PS3 encodes a thermotolerant heme O synthase; Saiki K et al.; The cyoE gene of the Escherichia coli bo-type quinol oxidase operon (cyoABCDE) has been previously shown to encode heme O synthase . To demonstrate a catalytic role of a cyoE homologue (the caaE gene) in the gene cluster for caa3-type cytochrome c oxidase of thermophilic Bacillus PS3, we have carried out genetic complementation analysis using the chimeric operon cyoABCD-caaE and heme O synthase assay using the CaaE-overproduced E . coli membranes . We found that the caaE gene encodes a thermotolerant heme O synthase which provides an intermediate for heme A biosynthesis. FEBS Lett, 1994 Sep 5, 351(2), 241 - 2 Alterations at the 3'-CCA end of Escherichia coli and Thermus thermophilus tRNA(Phe) do not abolish their acceptor activity; Moor NA et al.; The 3'-CCA end of tRNA(Phe) from Escherichia coli and Thermus thermophilus was changed to AAA, CCC, UUU and UUA by the stepwise degradation procedure of the 3'-CCA end of tRNA(Phe) followed by the ligation with oligoribonucleotides . Substrate activity of tRNA(UUAPhe) and tRNA(CCCPhe) in tRNA aminoacylation was shown . tRNA(AAAPhe) is a bad substrate for E . coli and Th . thermophilus phenylalanyl-tRNA synthetases . tRNA(UUUPhe) has no detectable activity in tRNA aminoacylation . Therefore the nature of the 3'-end of tRNA(Phe) plays an important role in tRNA binding and its substrate efficiency . Nevertheless the CCA sequence at the 3'-end of tRNA(Phe) does not seem to be an absolute requirement for tRNA aminoacylation. J Mol Biol, 1994 Sep 2, 241(5), 732 - 5 Crystallisation of the glycyl-tRNA synthetase from Thermus thermophilus and initial crystallographic data; Logan DT et al.; The glycyl-tRNA synthetase from Thermus thermophilus is a dimer of molecular mass 115 kDa, which has been crystallised using the vapour diffusion method from 5 to 7% polyethylene glycol 6000, 0.8 to 1.4 M NaCl at protein concentrations of 2 to 8 mg/ml . Nucleation is carried out at 4 degrees C and crystals are subsequently transferred to 15 degrees C to maximise growth . Crystals are truncated rhombohedra measuring on average 0.4 mm x 0.4 mm x 0.2 mm, which appear within a few days and reach full size in one to two months . GlyRS crystallises in two closely related space groups, P2(1)2(1)2(1) and C2,2,2(1), both with the same cell a = 125 A, b = 254 A, c = 104 A . Crystal packing in P2(1)2(1)2(1) is strongly C-centred . The crystals have VM = 3.6 A3/Da and a solvent content of 61%, with one dimer in the asymmetric unit in C2,2,2(1) and two dimers in P2(1)2(1)2(1) . The best native data extend to 2.9 A in C2,2,2(1) and are 90.6% complete with an R-factor between symmetry-related reflections of 10.0% . The structure has been solved by multiple isomorphous replacement and model building is in progress. J Biol Chem, 1994 Sep 2, 269(35), 22169 - 72 Tetrahymena ribozyme disrupts rRNA processing in yeast; Good L et al.; The intervening sequence (IVS) of Tetrahymena thermophila nucleolar DNA interrupts a highly conserved sequence in the RNA core structure of the large ribosomal subunit . This location in nuclear DNA is unusual as most group I introns are in mitochondrial and chloroplast DNA . To examine the effect of a ribozyme insertion in another nuclear genome, the Tetrahymena IVS was introduced into the analogous position in a cloned Schizosaccharomyces pombe ribosomal gene, and the mutant rDNA was expressed in vivo . RNA analyses indicated that mature 5.8 S rRNA was not formed from the mutant gene transcript and the amount of 27 S nRNA was significantly reduced . In contrast, hybridization analyses indicated that RNA splicing continued, and normal forms of free ribozyme were present . The results show that the IVS sequence can interfere with rRNA processing and suggest that the unusual amplification of a single rDNA repeat may have forced Tetrahymena to accommodate its ribozyme. J Biol Chem, 1994 Sep 2, 269(35), 22021 - 6 Divalent metal ion requirements of a thermostable multimetal beta-galactosidase from Saccharopolyspora rectivirgula; Harada M et al.; To understand the roles of metal ions on the catalytic properties and thermostability of the thermostable beta-galactosidase of Saccharopolyspora rectivirgula, a thermophilic actinomycete, we have investigated the binding kinetics and requirements of divalent metal ions by equilibrium dialysis, titration, and metal ion buffer techniques . We found that the monomeric multimetal enzyme (M(r) 136,977) had eight specific binding sites for divalent metal ions . These sites were classified as follows: a very tight (class I) site for Ca2+, three tight (class II) sites consisting of two Ca(2+)-specific sites (class IICa) and one Mn(2+)-specific site (class IIMn; Kd for Mn2+, 2.0 nM), and four loose (class III) sites for Mn2+ (Kd, 1.2 microM) and Mg2+ (Kd, 2 microM) . Removal of metal ions bound to class II and III sites of the holoenzyme (Ca3Mn5 species; relative Vmax (Vrel), 100%) by a chelating resin at 4 degrees C yielded a less thermostable Ca1 species (Vrel, 1.7%) with a class I Ca2+ ion, removal of which by a chelating resin at 50 degrees C caused a complete irreversible inactivation of the enzyme . Titration studies revealed that stoichiometric binding of Mn2+ to a class IIMn site of the Ca1 species caused a 33-fold activation whereas binding of Ca2+ to class IICa sites had no effect on enzyme activity . Ca1 species could be also activated 8-fold by heating at 60 degrees C for 20 min, suggesting that the catalytically important class II Mn2+ plays important roles in maintaining the native structure essential for activity . Occupation of class III sites by Mg2+ or Mn2+ was of physiological importance to attain sufficient thermostability by which this extracellular beta-galactosidase remained active for a prolonged time at elevated temperatures as was observed during growth of S . rectivirgula. Arch Biochem Biophys, 1994 Sep, 313(2), 280 - 6 The hyperthermophilic glycolytic enzyme enolase in the archaeon, Pyrococcus furiosus: comparison with mesophilic enolases; Peak MJ et al.; High enolase activity, as measured by the conversion of 2-phosphoglycerate to phosphoenolpyruvate, was found in the cytoplasm of Pyrococcus furiosus (an anaerobic, hyperthermophilic archaeon that grows optimally at 100 degrees C) . In this organism, the enzyme probably functions in a sugar fermentation pathway . The enzyme was purified to homogeneity . It had a temperature optimum of > 90 degrees C and a pH optimum of 8.1 . The enzyme was extremely thermostable with a time for 50% inactivation at 100 degrees C of 40 min . In contrast, an enolase from yeast was totally inactivated in 1 min at 88 degrees C . Both the P . furiosus and yeast enzymes required a metal ion for activity, but whereas the yeast enzyme has an absolute requirement for Mg2+, the P . furiosus enolase was equally active in the presence of Mn2+ . Both enzymes were competitively inhibited by citrate . P . furiosus enolase, as for mesophilic enolases, probably has a homodimeric structure with subunit M(r) greater than 45,000 . A highly conserved sequence of eight amino acids in the N-terminal region was found in enolases from P . furiosus and a wide range of other organisms including bacteria, yeast, birds, and mammals . Substantial differences in the thermal properties of the hyperthermophilic enzyme compared with that from less extreme thermophiles and mesophiles might be due to a substantially enhanced composition of hydrophobic amino acids. Mol Cell Biol, 1994 Sep, 14(9), 5939 - 49 A small family of elements with long inverted repeats is located near sites of developmentally regulated DNA rearrangement in Tetrahymena thermophila; Wells JM et al.; Extensive DNA rearrangement occurs during the development of the somatic macronucleus from the germ line micronucleus in ciliated protozoans . The micronuclear junctions and the macronuclear product of a developmentally regulated DNA rearrangement in Tetrahymena thermophila, Tlr1, have been cloned . The intrachromosomal rearrangement joins sequences that are separated by more than 13 kb in the micronucleus with the elimination of moderately repeated micronucleus-specific DNA sequences . There is a long, 825-bp, inverted repeat near the micronuclear junctions . The inverted repeat contains two different 19-bp tandem repeats . The 19-bp repeats are associated with each other and with DNA rearrangements at seven locations in the micronuclear genome . Southern blot analysis is consistent with the occurrence of the 19-bp repeats within pairs of larger repeated sequences . Another family member was isolated . The 19-mers in that clone are also in close proximity to a rearrangement junction . We propose that the 19-mers define a small family of developmentally regulated DNA rearrangements having elements with long inverted repeats near the junction sites . We discuss the possibility that transposable elements evolve by capture of molecular machinery required for essential cellular functions. J Appl Bacteriol, 1994 Sep, 77(3), 288 - 95 Aminopeptidase N from Streptococcus salivarius subsp . thermophilus NCDO 573: purification and properties; Midwinter RG et al.; A 96 kDa aminopeptidase was purified from Streptococcus salivarius subsp . thermophilus NCDO 573 . The enzyme had similar properties to aminopeptidases isolated from lactococci and lactobacilli and showed a high degree of N-terminal amino acid sequence homology to aminopeptidase N from Lactococcus lactis subsp . cremoris . It catalysed the hydrolysis of a range of aminoacyl 4-nitroanilides and 7-amido-4-methylcoumarin derivatives, dipeptides, tripeptides and oligopeptides . In common with aminopeptidases from other lactic acid bacteria, the enzyme from Strep . salivarius subsp . thermophilus showed highest activity with lysyl derivatives but was also very active with arginyl and leucyl derivatives . Relative activity with alanyl, phenylalanyl, tyrosyl, seryl and valyl derivatives was considerably lower and with glycyl, glutamyl and prolyl derivatives almost negligible . The aminopeptidase also catalysed the hydrolysis of dipeptides and tripeptides but mostly at rates much less than that with L-lysyl-4-nitroanilide and oligopeptides . The enzyme catalysed the successive hydrolysis of various amino acid residues from the N-terminus of several oligopeptides but it was unable to cleave peptide bonds on the N-terminal side of a proline residue. Cell Biochem Funct, 1994 Sep, 12(3), 221 - 6 A calcium-dependent protein kinase is present in tetrahymena; Hegyesi H et al.; A Ca(2+)-dependent protein kinase of Tetrahymena thermophila has been partially purified and characterized . The molecular mass of the enzyme is less than that of similar enzymes (for example protein kinase C), being about 55 kDa . After purification and in the presence of Ca2+ the enzyme activity increased . The promoter of protein kinase C (PKC) activity, phorbol myristate acetate (PMA), increased the activity while the protein kinase inhibitor H-7 decreased the activity of the enzyme . The experiments demonstrate the presence, activity and similarity to vertebrate enzymes of a protein kinase at a low level of phylogeny. Eur J Biochem, 1994 Sep 1, 224(2), 497 - 506 Gene cloning and characterization of PepC, a cysteine aminopeptidase from Streptococcus thermophilus, with sequence similarity to the eucaryotic bleomycin hydrolase; Chapot-Chartier MP et al.; Streptococcus thermophilus CNRZ 302 contains at least three general aminopeptidases able to hydrolyze Phe-beta-naphthylamide substrate . The gene encoding one of these aminopeptidases was cloned from a total DNA library of S . thermophilus CNRZ 302 constructed in Escherichia coli TG1 using pBluescript plasmid . The wild-type TG1 strain, although not deficient in aminopeptidase activity, is unable to hydrolyze the substrate Phe-beta-naphthylamide, and thus the library could be screened with an enzymic plate assay using this substrate . One clone was selected which was shown to express an aminopeptidase, identified as a PepC-like enzyme on the basis of cross-reactivity with polyclonal antibodies directed against the lactococcal PepC cysteine aminopeptidase . The gene was further subcloned and sequenced . A complete open reading frame coding for a 445-residue (50414 Da) polypeptide was identified . 70% identity was found between the deduced amino acid sequence and the sequence of PepC from Lactococcus lactis subspecies cremoris, confirming the identity of the cloned gene . High sequence similarity (38% identity) was also found with an eucaryotic enzyme, bleomycin hydrolase . In addition, the predicted amino acid sequence of the streptococcal PepC showed a region of strong similarity to the active site of cysteine proteinases with conservation of the residues involved in the catalytic site . The product of the cloned pepC gene was overproduced in E . coli and was purified from a cellular extract . Purification to homogeneity was achieved by two-step ion-exchange chromatography . Biochemical characterization of the pure recombinant enzyme confirms that the cloned peptidase is a thiol aminopeptidase possessing a broad specificity . The enzyme has a molecular mass of 300 kDa suggesting an hexameric structure . On the basis of sequence similarities as well as common biochemical and enzymic properties, the bacterial PepC-type enzymes and the eucaryotic bleomycin hydrolase constitute a new family of thiol aminopeptidases among the cysteine peptidases. Pathol Biol (Paris), 1994 Sep, 42(7), 706 - 10 {Links between risks of aspergillosis and environmental contamination . Review of the literature}; Nolard N; An increasing number of nosocomial invasive aspergilloses is related with the development of new therapies and to Aspergillus spores in the vicinity of the patient . The infiltration of thermophilic fungal spores in the patients' environment is linked not only with contaminated air systems but also to different other factors among which earth moving, demolition or renovation works adjacent to or inside the hospital . Potted plants and individual sachets of ground pepper distributed to patients are another source of Aspergillus spores . To limit fungal exposure, patients undergoing bone marrow transplant should be isolated under laminar flow while simple procedures such as taping windows and doors, shutting down air systems and mycological environmental control measures should be encouraged to protect immunocompromised patients. Biol Pharm Bull, 1994 Sep, 17(9), 1171 - 5 Studies on thermophile products . IX . Isofatty acid-containing phosphatidylglycerol that enhances the induction of concanavalin A-activated suppressor T cells; Kohama Y et al.; A new enhancer of the induction of concanavalin A (Con A)-activated suppressor T (Ts) cells has been demonstrated in the ethanolysate of Bacillus stearothermophilus UBT8038 . It was purified by successive silica gel column chromatographies and identified as phosphatidylglycerol with C14:0-C18:0 isofatty acids (Fr . 7-C) . Mouse splenocytes activated with Con A and Fr . 7-C (0.01-1 microgram/ml) in vitro significantly suppressed the proliferative response of syngenic splenocytes by mitogen stimulation in a dose-dependent manner, compared to those stimulated by Con A alone . The immunosuppressive response enhanced by Fr . 7-C disappeared when the cell populations of Thy-1.2 or CD8 positive lymphocytes were depleted . The result strongly suggests that Fr . 7-C is an immunosuppressive substance which enhances the induction of Con A-activated CD8 positive Ts cells. Mikrobiol Z, 1994 Sep-Oct, 56(5), 8 - 16 {The serine proteinases of thermophilic bacilli}; Pavlova IN et al.; The method of affinity chromatography using bacitracine as a specific ligand was applied to isolate and purify serine proteinases of 8 strains of thermophilic bacilli, including 4 strains of the species Bacillus subtilis, 2 strains of the species B . licheniformis and 2 strains of the species B . circulans . Study of physicochemical and catalytic properties of the enzymes of thermophiles has shown their complete identity with subtilysines as to molecular weight, optimal action conditions, sensitivity to inhibitors and specificity of the action both in respect to natural and in respect to artificial substrates of subtilysines . Four of eight studied enzymes (two of B . licheniformis, two of B . subtilis) were serologically relative to subtilysines of the type of Karlsberg; the rest of the enzymes did not produce the precipitation lines in the reaction of double immunodiffusion in gel with antiserum to subtilysine of the Karlsberg type . All the studied enzymes differed from commercial preparations of subtilysines (producers: B . licheniformis and B . subtilis) in the higher thermal stability: time of half-inactivation at 60 degrees C was 4.5-6 h for the enzymes from different strains of thermophiles; at 80 degrees C this value was 8-13 min . A capacity to lyse the living cells of some gram-negative bacteria and yeast is the most characteristic feature of the studied proteinases of thermophiles as against analogous enzymes of mesophilic strains of bacilli and enzymes of the animal origin. Protein Sci, 1994 Sep, 3(9), 1436 - 43 The chaperonin from the archaeon Sulfolobus solfataricus promotes correct refolding and prevents thermal denaturation in vitro; Guagliardi A et al.; We have isolated a chaperonin from the hyperthermophilic archaeon Sulfolobus solfataricus based on its ability to inhibit the spontaneous refolding at 50 degrees C of dimeric S . solfataricus malic enzyme . The chaperonin, a 920-kDa oligomer of 57-kDa subunits, displays a potassium-dependent ATPase activity with an optimum temperature at 80 degrees C . S . solfataricus chaperonin promotes correct refoldings of several guanidine hydrochloride-denatured enzymes from thermophilic and mesophilic sources . At a molar ratio of chaperonin oligomer to single polypeptide chain of 1:1, S . solfataricus chaperonin completely inhibits spontaneous refoldings and suppresses aggregation upon dilution of the denaturant; refoldings resume upon ATP hydrolysis, with yields of active molecules and rates of folding notably higher than in spontaneous processes . S . solfataricus chaperonin prevents the irreversible inactivations at 90 degrees C of several thermophilic enzymes by the binding of the denaturation intermediate; the time-courses of inactivations are unaffected and most activity is regained upon hydrolysis of ATP . S . solfataricus chaperonin completely prevents the formation of aggregates during thermal inactivation of chicken egg white lysozyme at 70 degrees C, without affecting the rate of activity loss; ATP hydrolysis results in the recovery of most lytic activity . Tryptophan fluorescence measurements provide evidence that S . solfataricus chaperonin undergoes a dramatic conformational rearrangement in the presence of ATP/Mg, and that the hydrolysis of ATP is not required for the conformational change . The ATP/Mg-induced conformation of the chaperonin is fully unable to bind the protein substrates, probably due to disappearance or modification of the substrate binding sites . This is the first archaeal chaperonin whose involvement in protein folding has been demonstrated. Biokhimiia, 1994 Sep, 59(9), 1299 - 303 {The role of the 3'-CCA sequence in the interaction of tRNA(Phe) from E . coli and Thermus thermophilus with homologous phenylalanyl-tRNA-synthetases}; Moor NA et al.; The 3'-CCA end of tRNA(Phe) from E . coli and Thermus thermophilus was modified by stepwise degradation and ligation of the shortened tRNA with different trinucleotides (pUpUpA, (pA)3, (pC)3, (pU)3) . Kinetic parameters for the aminoacylation reaction of modified tRNAs have been determ