Protein Expr Purif, 1995 Oct, 6(5), 707 - 15 High yield purification of a four subunit caa3-type cytochrome oxidase from the thermophilic bacterium Bacillus PS3 using fast protein liquid chromatography; Kirken RA et al.; The thermophilic bacterium, Bacillus PS3, was grown in a vigorously aerated nutrient broth at 65 degrees C with 100 mM glutamic acid serving as a supplemental carbon and nitrogen source . These growth conditions resulted in membranes highly enriched in cytochrome c oxidase (COX) {23.32 +/- 4.32 nmol heme a/g of cells (n = 5)}, which is nearly a threefold higher concentration of COX (heme caa3-type) than previously reported for this organism . A new high-yield purification of COX was performed by extracting the bacterial membranes with Triton X-100 (7 mg/mg protein), followed by ion-exchange fast liquid protein chromatography using a QAE (trimethyl ammonium) resin with subsequent hydroxyapatite chromatography and ammonium sulfate fractionation . This purification regime resulted in a 16% yield of cytochrome c oxidase with 20 mg of pure caa3-type COX (13 nmol heme a/mg protein) isolated from 100 g of cells . SDS-PAGE showed that the isolated enzyme had four subunits with apparent Mr of 68, 38, 23, and 13 kDa . In addition, a new 34-kDa peptide was also detected in this preparation, which may represent the ORF1 gene product for this organism . Subunit II (Mr = 38 kDa) of the isolated enzyme was shown to contain covalently bound heme c by using both heme-staining of SDS-PAGE and immunoreactivity with an anti-cytochrome c antibody . The purified enzyme also exhibited high electron transfer activity (340s-1) when assayed at pH 6.5 in the presence of the nonionic detergent, beta-dodecyl maltoside. Protein Expr Purif, 1995 Oct, 6(5), 637 - 45 Overexpression and purification of Thermus thermophilus elongation factors G, Tu, and Ts from Escherichia coli; Blank J et al.; The translation elongation factors G (EF-G), Tu (EF-Tu), and Ts (EF-Ts) from the extreme thermophilic bacterium Thermus thermophilus were overproduced in Escherichia coli . The fus gene coding for EF-G and the tufA gene coding for EF-Tu were expressed under the control of a tac promoter, whereas EF-Ts was overproduced with the T7 RNA polymerase system . A detailed description for the purification of the three elongation factors from E . coli is presented . EF-G and EF-Tu are isolated by Q-Sepharose FF chromatography, heat treatment at 65 or 60 degrees C, respectively, and Sephacryl S200 gel permeation chromatography . For the purification of EF-Ts, a heat denaturation step is followed by DEAE-cellulose chromatography and a cation exchange EMD-SO-3 650 column . The overproduced factors show the same properties as those purified from T . thermophilus . As the crystal structures of T . thermophilus EF-Tu and EF-G have been solved recently, many questions concerning the function of particular residues or domains arise, which may be best addressed by studying the in vitro behavior and structure of altered recombinant constructs . The methods presented here should facilitate such studies. J Appl Bacteriol, 1995 Oct, 79(4), 447 - 53 Purification and characterization of the major beta-1,4-endoglucanase from Thermomonospora curvata; Lin SB et al.; The major beta-1,4-endoglucanase (EG) of the thermophilic actinomycete, Thermomonospora curvata, contributed over 80% of the total EG activity recovered from cell-free culture fluid after growth on cellulose . The enzyme was purified to electrophoretic homogeneity by ammonium sulphate precipitation, ion-exchange chromatography and size exclusion HPLC . This monomeric enzyme had a specific activity of 750 IU mg(-1) when assayed with 2.5% (w/v) carboxymethyl cellulose (CMC) at 70 degrees C, pH 6.0 . Highest activity was observed on CMC with a degree of polymerization of 3200 . The EG was stable for 48 h at 60 degrees C, pH 6.0 and had a half-life of 30 min at 80 degrees C; temperature and pH optima were 70-73 degrees C and 6.0-6.5, respectively . The mol . wt was 100,000 and the pI was 4.0 . The Km and Vmax values were 7.33 mg/ml(-1) and 833 microns min(-1), respectively . EG activity was inhibited by Fe(2+), Hg(2+), Ag(+) and Pb(2+), and enhanced by dithiothreitol and Zn(2+) . The first 12 amino acid residues at the N-terminus were: Asp-Glu-Val-Asp-Glu-Ile-Arg-Asn-Gly-Asp-Phe-Ser . Glutamic and aspartic acid constituted 24% of the total amino acid composition; no amino sugar was found. Eur J Biochem, 1995 Oct 1, 233(1), 227 - 37 Dimeric 3-phosphoglycerate kinases from hyperthermophilic Archaea . Cloning, sequencing and expression of the 3-phosphoglycerate kinase gene of Pyrococcus woesei in Escherichia coli and characterization of the protein . Structural and functional comparison with the 3-phosphoglycerate kinase of Methanothermus fervidus; Hess D et al.; The gene coding for the 3-phosphoglycerate kinase (EC 2.7.2.3) of Pyrococcus woesei was cloned and sequenced . The gene sequence comprises 1230 bp coding for a polypeptide with the theoretical M(r) of 46,195 . The deduced protein sequence exhibits a high similarity (46.1% and 46.6% identity) to the other known archaeal 3-phosphoglycerate kinases of Methanobacterium bryantii and Methanothermus fervidus {Fabry, S., Heppner, P., Dietmaier, W . & Hensel, R . (1990) Gene 91, 19-25} . By comparing the 3-phosphoglycerate kinase sequences of the mesophilic and the two thermophilic Archaea, trends in thermoadaptation were confirmed that could be deduced from comparisons of glyceraldehyde-3-phosphate dehydrogenase sequences from the same organisms {Zwickl, P., Fabry, S., Bogedain, C., Haas, A . & Hensel, R . (1990) J . Bacteriol . 172, 4329-4338} . With increasing temperature the average hydrophobicity and the portion of aromatic residues increases, whereas the chain flexibility as well as the content in chemically labile residues (Asn, Cys) decreases . To study the phenotypic properties of the 3-phosphoglycerate kinases from thermophilic Archaea in more detail, the 3-phosphoglycerate kinase genes from P . woesei and M . fervidus were expressed in Escherichia coli . Comparisons of kinetic and molecular properties of the enzymes from the original organisms and from E . coli indicate that the proteins expressed in the mesophilic host are folded correctly . Besides their higher thermostability according to their origin from hyperthermophilic organisms, both enzymes differ from their bacterial and eucaryotic homologues mainly in two respects . (a) The 3-phosphoglycerate kinases from P . woesei and M . fervidus are homomeric dimers in their native state contrary to all other known 3-phosphoglycerate kinases, which are monomers including the enzyme from the mesophilic Archaeum M . bryantii . (b) Monovalent cations are essential for the activity of both archaeal enzymes with K+ being significantly more efficient than Na+ . For the P . woesei enzyme, non-cooperative K+ binding with an apparent Kd (K+) of 88 mM could be determined by kinetic analysis, whereas for the M . fervidus 3-phosphoglycerate kinase the K+ binding is rather complex: from the fitting of the saturation data, non-cooperative binding sites with low selectivity for K+ and Na+ (apparent Kd = 270 mM) and at least three cooperative and highly specific K+ binding sites/subunit are deduced . At the optimum growth temperature of P . woesei (100 degrees C) and M . fervidus (83 degrees C), the 3-phosphoglycerate kinases show half-lives of inactivation of only 28 min and 44 min, respectively.(ABSTRACT TRUNCATED AT 400 WORDS) Appl Microbiol Biotechnol, 1995 Oct, 43(5), 856 - 60 Debranching of arabinoxylan: properties of the thermoactive recombinant alpha-L-arabinofuranosidase from Clostridium stercorarium (ArfB); Schwarz WH et al.; The gene arfB encoding alpha-L-arabinofuranosidase B of the cellulolytic thermophile Clostridium stercorarium was expressed in Escherichia coli from a 2.2-kb EcoRI DNA fragment . The recombinant gene product ArfB was purified by fast-performance liquid chromatography . It has a tetrameric structure with a monomeric relative molecular mass of 5200 . The optima for temperature and pH are 70 degrees C and 5.0 respectively . The enzyme appears to have no metal cofactor requirement and is sensitive to sulfhydryl reagents . It hydrolyzes aryl and alkyl alpha-L-arabinofuranosides and cleaves arabinosyl side-chains from arabinoxylan (oat-spelt xylan) and from xylooligosaccharides produced by recombinant endoxylanase XynA from the same organism . The identify of the N-terminal amino acid sequences indicates that ArfB corresponds to the major alpha-arabinosidase activity present in the culture supernatant of C . stercorarium. Biotechnol Appl Biochem, 1995 Oct, 22 ( Pt 2), 203 - 14 Enzyme thermostabilization by bovine serum albumin and other proteins: evidence for hydrophobic interactions; Chang BS et al.; BSA stabilizes Streptococcus thermophilus beta-galactosidase against thermal inactivation and binds to the active enzyme subunits formed on heating . The mechanism of interaction and stabilization, however, is unknown, and it was investigated using different proteins . The results show that several proteins increased the enzyme half-life (t 1/2) at 64 degrees C in the presence of the substrate lactose . The best stabilizers were BSA (9-fold) and casein (6-fold) . There was a significant correlation between enzyme half-life (t 1/2) and surface hydrophobicity of the proteins (So), of the form t 1/2 varies; is directly proportional to S0.5o . The surface hydrophobicity of the enzyme increased upon heating, while that of BSA declined . Heating enzyme and BSA together caused a net loss in surface hydrophobicity, indicating hydrophobic interactions, but there was no change in the absence of heating . Stabilization of the enzyme by BSA was markedly affected by chaotropic and kosmotropic salts . Stabilization was increased by 1 M Na2SO4 and reduced by 1 M NaBr; 1 M NaCl had little effect . None of the three salts increased the stability of the enzyme itself, indicating that the effect was on the enzyme-protein interaction . The results indicate that BSA stabilized the enzyme by hydrophobic interactions with the heated enzyme and that surface hydrophobicity is a major determinant of the extent of stabilization by a protein. Virology, 1995 Oct 1, 212(2), 632 - 40 Characterization of a temperate Streptococcus thermophilus bacteriophage and its genetic relationship with lytic phages; Brussow H et al.; The temperate Streptococcus thermophilus bacteriophage phi SFi21 showed an 38-kb-long double-stranded DNA genome with cohesive ends . A single integration site was used in lysogens established in three different S . thermophilus strains . The attP and attB sites were localized on the restriction map of phage DNA and by hybridization on pulsed field separated bacterial DNA . All laboratory-established lysogens showed in addition to integrated prophage DNA unintegrated monomer phage DNA with unligated cos sites . The genetic relatedness of phi SFi21 DNA with DNA from lytic phages was studied in dot blot and Southern blot hybridization by using individual restriction fragments of phiSFi21 DNA as probes . Lytic group I phages hybridized with fragments of the central and the right part of the phiSFi21 genome but failed to hybridize with a fragment joining both parts . Lytic group II phages showed hybridization with the right half of the phiSFi21 genome . In lytic group IV phages, biologically a heterogeneous group, many different combinations of cross hybridization were detected in accordance with the hypothesis of the modular evolution of phage genomes. J Bacteriol, 1995 Oct, 177(19), 5473 - 9 Motility and thermotactic responses of Thermotoga maritima; Gluch MF et al.; Thermotoga maritima, a thermophilic eubacterium, is motile at temperatures ranging from 50 to 105 degrees C . The cells are propelled by a single flagellum which most of the time spins clockwise . Changes in the swimming direction ("tumbles") are achieved by short reversals of the direction of filament rotation . The average speed of swimming cells depends on the temperature, reaching a maximum value of about 60 microns/s at 85 degrees C . The cells show a thermotactic response to temporal temperature changes . When the temperature is raised, the rate of tumbles is increased, while decreasing temperature decreases the tumbling rate. J Bacteriol, 1995 Oct, 177(19), 5460 - 6 Horizontal transference of S-layer genes within Thermus thermophilus; Fernandez-Herrero LA et al.; The S-layers of Thermus thermophilus HB27 and T . thermophilus HB8 are composed of protein units of 95 kDa (P95) and 100 kDa (P100), respectively . We have selected S-layer deletion mutants from both strains by complete replacement of the slpA gene . Mutants of the two strains showed similar defects in growth and morphology and overproduced an external cell envelope inside of which cells remained after division . However, the nature of this external layer is strain specific, being easily stained and regular in the HB8 delta slpA derivative and amorphous and poorly stained in the HB27 delta slpA strain . The addition of chromosomic DNA from T . thermophilus HB8 to growing cultures of T . thermophilus HB27 delta slpA led to the selection of a new strain, HB27C8, which expressed a functional S-layer composed of the P100 protein . Conversely, the addition of chromosomic DNA from T . thermophilus HB27 to growing cultures of T . thermophilus HB8 delta slpA allowed the isolation of strain HB8C27, which expressed a functional S-layer composed of the P95 protein . The driving force which selected the transference of the S-layer genes in these experiments was the difference in growth rates, one of the main factors leading to selection in natural environments. Dev Biol, 1995 Oct, 171(2), 497 - 506 The effects of lithium chloride on pattern formation in Tetrahymena thermophila; Jerka-Dziadosz M et al.; Lithium ions have long been known to exert dramatic effects on the specification of cell fates in multicellular systems . We have analyzed the effects of Li+ on intracellular patterning in a complex unicellular organism, the ciliate Tetrahymena thermophila . LiCl does not affect the locations of major structural landmarks in the cortical region of wild-type cells and does not modify the phenotype of pattern-mutant cells . However, in all strains studied LiCl differentially affects early stages of oral development . It initially triggers a slow regression of oral primordia, which is followed by an excessive proliferation of basal bodies that leads to a hypertrophy of the ciliature of the cell's feeding organelle . This hypertrophy mimics the effects of the membranellar-pattern-D mutation, the phenotype of which is enhanced in the presence of LiCl . These effects were partially reversed by myo-inositol; however, neomycin failed to mimic the effects of LiCl . Thus, although lithium ions have major cellular effects on Tetrahymena, they do not influence the specification of the body plan in a manner analogous to that observed in multicellular organisms and may work in part through mechanisms other than the now-classical inositol-phosphate cycle. Int J Syst Bacteriol, 1995 Oct, 45(4), 811 - 9 Comparison of Mycobacterium 23S rRNA sequences by high-temperature reverse transcription and PCR; Stone BB et al.; We describe a modified rRNA sequence analysis method which we used to determine the phylogenetic relationships among 58 species belonging to the genus Mycobacterium . We combined the sensitivity of the reverse transcriptase PCR for amplifying nanogram amounts of template rRNA material with the elevated extension temperatures used for the thermostable DNA polymerase from Thermus thermophilus . A 70 degrees C reverse transcription extension step permitted improved read-through of highly structured rRNA templates from members of the genus Mycobacterium, which have G+C contents of 66 to 71 mol% . The nucleic acid sequences of the amplified material were then determined by performing thermal cycle sequencing with alpha-33P-labeled primers, again with extension at 70 degrees C . Nonspecifically terminated bands were chased by using terminal deoxynucleotidyl transferase . Our method had a template requirement of nanogram amounts or less of purified RNA or 2,000 CFU of intact cells and had sufficient sensitivity so that lyophils obtained from the American Type Culture Collection could be used as source material . Sequences from a 250-nucleotide stretch of the 23S rRNA were aligned, and phylogenetic trees were evaluated by using the De Soete distance treeing algorithm and Rhodococcus bronchialis as the outgroup . Our 23S rRNA trees were compared with previously published 16S rRNA trees, including the comprehensive trees developed by the University of Illinois Ribosomal Database Project, and included 15 species not evaluated previously . Most of the groups were in general agreement and were consistent with relationships determined on the basis of biochemical characteristics, but some new relationships were also observed. Int J Syst Bacteriol, 1995 Oct, 45(4), 783 - 9 Description of Thermoanaerobacter brockii subsp . lactiethylicus subsp . nov., isolated from a deep subsurface French oil well, a proposal to reclassify Thermoanaerobacter finnii as Thermoanaerobacter brockii subsp . finnii comb . nov., and an emended description of Thermoanaerobacter brockii; Cayol JL et al.; A strictly anaerobic, thermophilic, gram-positive, spore-forming cubacterium designated strain SERB 5268T (T = type strain) was isolated from an oil field at a depth of 2,100 m, where the temperature was 92 degrees C . The cells of this organism were gram-positive, straight, motile rods (0.5 by 2 to 3 microns) with peritrichous flagella . The cells occurred singly or in pairs during the logarithmic growth phase, but were pleomporphic and filamentous (length, 15 microns) in old cultures . Growth occurred at temperatures of 40 to 75 degrees C, and optimum growth occurred at temperatures between 55 and 60 degrees C . The fermentable substrates included glucose, fructose, galactose, mannose, cellobiose, maltose, sucrose, lactose, D-xylose, D-ribose, mannitol, pyruvate, and starch . The products of fermentation of glucose were lactate, acetate, ethanol, H2, and CO2 . The DNA base composition was 35 mol% G+C . The results of 16S rRNA sequence comparisons indicated that strain SEBR 5268T was closely related to Thermoanaerobacter brockii and Thermoanaerobacter finnii, and these three organisms exhibited levels of ribosomal DNA sequence homology of 98 to 99% . The results of DNA-DNA hybridization studies performed with the three organisms confirmed this close affiliation, and as base pairing values of > 70% were obtained, these organisms belong to the same species . Therefore, we propose that T . finnii should be reclassified as a subspecies of T . brockii, Thermoanaerobacter brockii subsp . finnii comb . nov . This automatically creates Thermoanaerobacter brockii subsp . brockii . We also propose that strain SEBR 5268T should be classified as a member of a new subspecies of T . brockii, Thermoanaerobacter brockii subsp . lactiehylicus.(ABSTRACT TRUNCATED AT 250 WORDS) Int J Syst Bacteriol, 1995 Oct, 45(4), 676 - 81 Chloroflexus aggregans sp . nov., a filamentous phototrophic bacterium which forms dense cell aggregates by active gliding movement; Hanada S et al.; Two strains of thermophilic photosynthetic bacteria, designated MD-66T (T = type strain) and YI-9, were isolated from bacterial mats in two separate hot springs in Japan . These new isolates were phenotypically similar to Chloroflexus aurantiacus in some respects . They were thermophilic filamentous photosynthetic bacteria that grew well at 55 degrees C either anaerobically as photoheterotrophs or aerobically as chemoheterotrophs . They exhibited gliding motility, produced bacteriochlorophylls a and c, contained chlorosomes, and required thiamine and folic acid as growth factors . However, isolates MD-66T and YI-9 had the ability to rapidly form mat-like dense aggregates of filaments, an ability which has not been observed in any C . aurantiacus strain . Carbon source utilization tests revealed that unlike C . aurantiacus, the new isolates did not utilize acetate, citrate, ethanol, or glycylglycine . An analysis of the carotenoid components revealed that isolates MD-66T and YI-9 contained mainly gamma-carotene and OH-gamma-carotene glucoside fatty acid esters . These isolates also contained only trace amounts of beta-carotene, which is a major carotenoid component (28.4% of the total carotenoids) in C . aurantiacus . The results of DNA hybridization studies suggested that the new strains were genetically distinct from C . aurantiacus (levels of similarity, 9 to 18%), and 16S rRNA sequence comparisons showed that strain MD-66T was related to C . aurantiacus at a similarity level of 92.8% . On the basis of our data, we propose that a new Chloroflexus species should be created for our new isolates; the name of this new species is Chloroflexus aggregans, and the type strain is strain MD-66 (= DSM 9485). Biochim Biophys Acta, 1995 Sep 27, 1252(1), 1 - 14 Understanding and increasing protein stability; Fagain CO; This review surveys the processes leading to loss of protein function and the types of molecular interaction that help stabilize proteins . It considers the effects of organic solvents on stability and the special features of thermophilic proteins . The deliberate manipulation of stability by protein engineering is discussed using the enzyme subtilisin as example . Both random and rational mutations of this protein have led to variants with greatly improved tolerances of high temperatures and organic solvents . One can also use chemical modification to modify protein stability and some of the main approaches are reviewed . The chemical and genetic strategies are complementary and have been combined to stabilize cytochrome c by metal-mediated cross-linking following site-specific mutagenesis . The article concludes by summarizing the beneficial effects of certain additives on protein stability. Gene, 1995 Sep 22, 163(1), 109 - 13 Cloning of a gene from Thermus filiformis and characterization of the thermostable nuclease; Fomenkov A et al.; A gene coding for a thermostable nuclease was cloned from the thermophilic microorganism, Thermus filiformis (Tf), using an indicator strain containing a dinD::lacZ fusion . The gene, designated nuc17, has been mapped within a 2300-bp fragment . The 55-kDa Tf nuclease was purified to over 95% homogeneity . Single-stranded (ss) DNA is the preferred substrate for the Tf nuclease, although double-stranded (ds) DNA can also be digested . Nuclease activity increases with increasing temperature up to 80 degrees C and requires the metal ions Ca++ or Mg++ for catalysis . Tf nuclease is primarily an endonuclease that leaves 5' phosphates in the digested products . The ssDNA extensions remaining after exonuclease III digestion of dsDNA can be removed by the Tf nuclease, making it a useful reagent to generate unidirectional deletions. Gene, 1995 Sep 22, 163(1), 103 - 7 A novel insertion sequence (IS)-like element of the thermophilic bacterium PS3 promotes expression of the alanine carrier protein-encoding gene; Murai N et al.; A novel insertion sequence (IS)-like element was found in the 5'-upstream region of the alanine carrier protein-encoding gene (acp) in the thermophilic bacterium PS3 chromosomal DNA . The sequence contained an open reading frame (ORF) encoding a polypeptide of 369 amino acids which revealed high similarity with ORFs from IS891 from the cyanobacterium Anabaena and IS1136 from Saccharopolyspora erythraea . The direction of transcription was the same as that of acp, and typical inverted and direct repeats characteristic of IS were found in both the 5' and 3' region of the ORF . Southern hybridization analysis of the chromosomal DNA revealed that multiple copies of the ORF sequence were contained in the PS3 genome . This element might well be a member of a new IS family including IS891 and IS1136, and we have designated this element IS1341 . The analysis of acp expression in Escherichia coli cells indicated that IS1341 promotes the expression of acp. J Biol Chem, 1995 Sep 15, 270(37), 22037 - 43 Affinity purification, overexpression, and characterization of chaperonin 10 homologues synthesized with and without N-terminal acetylation; Ryan MT et al.; Utilizing the ability of bacterial chaperonin 60 (GroEL) to functionally interact with chaperonin 10 (Cpn10) homologues in an ATP-dependent fashion, we have purified substantial amounts of mammalian, chloroplast, and thermophilic Cpn10 homologues from their natural host . In addition, large amounts of recombinant rat Cpn10 were produced in Escherichia coli and found to be identical to its authentic counterpart except for the lack of N-terminal acetylation . By comparing these two forms of Cpn10, it was found that acetylation does not influence the oligomeric structure of Cpn10 and is not essential for chaperone activity or mitochondrial import in vitro . In contrast, N-terminal acetylation proved crucial in the protection of Cpn10 against degradation by N-ethylmaleimide-sensitive proteases derived from organellar preparations of rat liver . The availability of large amounts of both affinity-purified and recombinant Cpn10 will facilitate not only further characterization of the eukaryotic folding machinery but also further scrutiny of the reported function of Cpn10 as early pregnancy factor. J Biol Chem, 1995 Sep 15, 270(37), 21571 - 8 ATP synthesis by the F0F1-ATPase from the thermophilic Bacillus PS3 co-reconstituted with bacteriorhodopsin into liposomes . Evidence for stimulation of ATP synthesis by ATP bound to a noncatalytic binding site; Richard P et al.; F-type ATPase from the thermophilic Bacillus PS3, TF0F1, which was essentially free of bound nucleotides after isolation and purification, was co-reconstituted into liposomes with the light-driven proton pump bacteriorhodopsin . The time course of the light-induced ATP synthesis was biphasic; an initial slow phase accelerated to a final steady-state rate two to three times faster . Adding ATP before initiating the reaction suppressed the slow phase, suggesting that the state of occupancy of specific sites by ATP regulated the synthetic activity of TF0F1 . Incubating the purified TF0F1 with ADP and ATP revealed one ADP and two ATP binding sites that were stable to gel filtration . We analyzed the time courses of light-induced ATP synthesis for the enzyme with different nucleotide content, after co-reconstitution into liposomes with bacteriorhodopsin . The two ATP sites were identified to have regulatory function . A complex containing TF0F1.ADP, 1:1, was co-reconstituted with various quantities of ATP to obtain a range of molar ratios of TF0F1.ADP:ATP of between 1:0 and 1:1.7 . It was found that the initial rate of ATP synthesis increased with the level of ATP bound to the enzyme . After binding one ATP, a stimulation of ATP synthesis by a factor of 2 was observed . The second ATP site also exhibited regulatory properties . It stimulated ATP synthesis but to a much smaller extent; the stimulation did not exceed 20% . Binding of the photoreactive analogues 2-azido-{alpha-32P}ADP and 2-azido-{alpha-32P}ATP to the TF0F1 and their effects on the rate of ATP synthesis are described further.(ABSTRACT TRUNCATED AT 250 WORDS) Biochem Biophys Res Commun, 1995 Sep 14, 214(2), 730 - 6 Gene of heat shock protein of sulfur-dependent archaeal hyperthermophile Desulfurococcus; Kagawa Y et al.; To elucidate thermoresistance, a gene of a hyperthermophilic heat shock protein (HHSP) was isolated from the hyperthermophile Desulfurococcus strain SY which grows at 95 degrees C . The molecular weight of HHSP deduced from the open reading frame was 59,137 (545 amino acid residues) . Sequence alignments of peptides reveal similarities (evolutionary distances) to the alpha (0.279) and beta (0.296) subunits of thermosome, TF55 (0.343) and human t-complex polypeptide 1 . The structure of a thermophilic heat shock protein TGroEL (Tamada et al . (1991) Biochem, Biophys . Res . Commun . 179, 565) was quite different from that of HHSP . TGroEL and HSP60 have sequences identical to HHSP at its equatorial domain, while those identical to the alpha subunit of F-type ATPase are at its apical domain. Biochem Biophys Res Commun, 1995 Sep 14, 214(2), 646 - 52 SaRD, a new protein isolated from the extremophile archaeon Sulfolobus acidocaldarius, is a thermostable ribonuclease with DNA-binding properties; Kulms D et al.; We have isolated the thermostable 9 kDa SaRD-protein from Sulfolobus acidocaldarius which exhibit RNase activity as well as DNA-binding properties (SaRD) . The amino acid composition and the sequence of the 16 N-terminal amino acids show similarities to different RNases as well as to DNA-binding proteins from thermophilic archea . The RNase activity was demonstrated by 5S rRNA degradation, thin layer chromatography and a zymogram . The temperature optimum for the RNase activity is 65 degrees C . The pH optimum ranges from 6.5-7.0 . DNA-binding properties were shown by gel-shift assays on agarose gels . In a similar way SaRD mediated protection of DNA against DNase I digestion and Sau3A I restriction could be demonstrated . The melting point (Tm) of genomic DNA was raised from 68 degrees C to 90 degrees C by addition of the SaRD-protein . CD spectroscopy indicated that SaRD is very stable near neutral pH and can neither be unfolded by temperatures up to 85% C nor by addition of 8 M urea. Biochim Biophys Acta, 1995 Sep 12, 1231(2), 139 - 46 Expression of the wild-type and the Cys-/Trp-less alpha 3 beta 3 gamma complex of thermophilic F1-ATPase in Escherichia coli; Matsui T et al.; The alpha, beta and gamma subunits of F1-ATPase from thermophilic Bacillus PS3 were expressed in Escherichia coli cells simultaneously in large amounts . Most of the expressed subunits assembled into a form of alpha 3 beta 3 gamma complex in E . coli cells and this complex was easily purified to homogeneity . The recombinant alpha 3 beta 3 gamma complex thus obtained showed similar enzymatic properties to the alpha 3 beta 3 gamma complex obtained by in vitro reconstitution from individual subunits (Yokoyama, K . et al . (1989) J . Biol . Chem . 264, 21837-21841) except that the former had several-fold higher ATPase activity than the latter . Using this expression system, a mutant alpha 3 beta 3 gamma complex with no Trp and Cys was generated by replacing alpha Cys193 and alpha Trp463 with Ser and Phe, respectively . This mutant complex was functionally intact, indicating both residues are not essential for catalysis . The Cys-/Trp-less complex is a convenient 'second wild type' enzyme from which one can generate mutants with Trp (as a fluorescent probe) or Cys (as an acceptor of a variety of probes) at desired positions without concern for 'background' Trp and Cys residues. Proc Natl Acad Sci U S A, 1995 Sep 12, 92(19), 8715 - 8 High frequency of sex and equal frequencies of mating types in natural populations of the ciliate Tetrahymena thermophila; Doerder FP et al.; In ciliate protists, sex involves the temporary joining of two cells of compatible mating type, followed by meiosis and exchange of gametic nuclei between conjugants . Reproduction is by asexual binary fission following conjugation . For the many ciliates with fixed multiple mating types, frequency-dependent sex-ratio theory predicts equal frequencies of mating types, if sex is common in nature . Here, we report that in natural populations of Tetrahymena thermophila sexually immature cells, indicative of recent conjugation, are found from spring through fall . In addition, the seven mating types occur in approximately equal frequencies, and these frequencies appear to be maintained by interaction between complex, multiple mat alleles and environmental conditions during conjugation . Such genotype-environment interaction determining mating type frequency is rare among ciliates. Biochim Biophys Acta, 1995 Sep 6, 1251(2), 170 - 6 Molecular properties of glutamate dehydrogenase from the extreme thermophilic archaebacterium Sulfolobus solfataricus; Facchiano AM et al.; This study is concerned with the structural characterization in solution of the glutamate dehydrogenase from the Archaeon Sulfolobus solfataricus . At neutral pH both alpha-helix and beta-sheet constitute the secondary structure of this enzyme, on the basis of circular dichroism . A complex, temperature dependent self-association equilibrium regulates the formation of the enzyme quaternary structure, which seems to be accompanied by a reversible structural change . At 25 degrees C the enzyme is mostly represented by monomeric subunits at concentrations lower than 0.02 mg/ml, while oligomers are predominant at concentrations higher than 0.12 mg/ml . The mid-point of the association curve shifts from 0.05 mg/ml at 25 degrees C to about 0.1 mg/ml at 45 degrees C . Only the oligomeric form appears to be temperature resistant . Monomeric and oligomeric enzyme show distinct behaviour on guanidine hydrochloride perturbation at neutral pH . The monomer denaturation, although complex, is reversible . Two fluorescent tryptophan classes are detectable in the monomer, monitoring the independent unfolding of two regions through a multistate transition . Instead, the oligomeric protein shows a complex denaturation pattern with the tendency to aggregate irreversibly at high denaturant concentration. Protein Eng, 1995 Sep, 8(9), 905 - 13 Composition analysis of alpha-helices in thermophilic organisms; Warren GL et al.; We present a statistical comparison of the amino acid composition in a secondary structure element, the alpha-helix, of proteins stable at high temperatures with those which are less so . This study has shown that the temperature-dependent Zimm-Bragg helix propagation value s is not a good predictor for the helix-forming tendency of an amino acid in thermostable proteins . However, we have shown that delta s, the change in s from 20 to 60 degrees C, accurately predicts the direction of the probability shift for 15 amino acids in thermostable protein alpha-helices, although it does not predict the magnitude of that change . The residues tyrosine, glycine and glutamine show a significant increase in residency in alpha-helices for thermostable proteins over their non-thermostable counterparts . Significant decreases in alpha-helix residency occur for the residues valine, glutamic acid, histidine, cysteine and aspartic acid in proteins from thermophilic organisms . Aromatic interactions, hydrogen bonding and a reduction of charge may explain the increase observed for tyrosine and glutamine and the decrease in glutamic acid and aspartic acid, although packing considerations cannot be ruled out . The only physical explanation for the increase in glycine would seem to be its positive delta s value. Rev Sci Tech, 1995 Sep, 14(3), 811 - 8 Reverse transcription combined with polymerase chain reaction as a detection method for pestiviral infections; Stadejek T et al.; An assay based on reverse transcription coupled with the polymerase chain reaction (RT-PCR) was used for the detection of hog cholera virus (HCV) and bovine virus diarrhoea virus (BVDV) in cell culture . In this study, a precipitate of the supernatants derived from cell cultures infected with HCV and BVDV was used in RT reactions, in place of extracted viral RNA . Both RT and PCR were performed using recombinant Thermus thermophilus (rTth) DNA polymerase . The specificity of the RT-PCR products was confirmed by hybridisation with a digoxygenin-labelled DNA probe . The results not only show that the stage of RNA isolation can be bypassed, but also illustrate an easy and efficient means of obtaining templates suitable for identification and characterisation of HCV and BVDV in tissue culture by RT-PCR. Hua Xi Yi Ke Da Xue Xue Bao, 1995 Sep, 26(3), 319 - 21 {Isolation and identification of extrem thermophilic bacteria from hot springs of Sichuan and Tibet}; Jia W et al.; Eleven strains of extrem thermophilic bacteria belonging to the Bacillus Stearothermophiles were isolated from the hot springs of Sichuan and Tibet . The cells were gram-positive, sporulating and motile . The optimum temperature for growth was between 65 degrees C to 70 degrees C; the maximum 94 degrees C, and minimum 40 degrees C . The colour of colony was yellow to bright orange . The G+C content of the DNA in strains has been found to be in the range 48.4-53.15 mol%. Biokhimiia, 1995 Sep, 60(9), 1435 - 49 {Site-specific endonuclease and methylase from thermophilic bacteria of Bacillus species IS4}; Zelinskaia NV et al.; The site-specific endonuclease R . BspIS4I and methylase M . BspIS4I have been isolated and purified to functional purity from the thermophilic strain of Bacillus species IS4 . R . BspIS4I recognizes sequence {sequence: see text} on the DNA and cleaves it as indicated by the arrows to form single-stranded 4-nucleotide 5'-protruding termini . The enzyme is an isoschizomer of BbvII . M . BspIS4I is related to adenine-specific methylase. J Bacteriol, 1995 Sep, 177(17), 4947 - 62 Structure of peptidoglycan from Thermus thermophilus HB8; Quintela JC et al.; The composition and structure of peptidoglycan (murein) extracted from the extreme thermophilic eubacterium Thermus thermophilus HB8 are presented . The structure of 29 muropeptides, accounting for more than 85% of total murein, is reported . The basic monomeric subunit consists of N-acetylglucosamine-N-acetylmuramic acid-L-Ala-D-Glu-L-Orn-D-Ala-D-Ala, acylated at the delta-NH2 group of Orn by a Gly-Gly dipeptide . In a significant proportion (about 23%) of total muropeptides, the N-terminal Gly is substituted by a residue of phenylacetic acid . This is the first time phenylacetic acid is described as a component of bacterial murein . Possible implications for murein physiology and biosynthesis are discussed . Murein cross-linking is mediated by D-Ala-Gly-Gly peptide cross-bridges . Glycan chains are apparently terminated by (1-->6) anhydro N-acetylmuramic acid residues . Neither reducing sugars nor murein-bound macromolecules were detected . Murein from T . thermophilus presents an intermediate complexity between those of gram-positive and gram-negative organisms . The murein composition and peptide cross-bridges of T . thermophilus are typical for a gram-positive bacterium . However, the murein content, degree of cross-linkage, and glycan chain length for T . thermophilus are closer to those for gram-negative organisms and could explain the gram-negative character of Thermus spp. J Biol Chem, 1995 Sep 1, 270(35), 20345 - 58 Molecular genetic and protein chemical characterization of the cytochrome ba3 from Thermus thermophilus HB8; Keightley JA et al.; Thermus thermophilus HB8 cells grown under reduced dioxygen tensions contain a substantially increased amount of heme A, much of which appears to be due to the presence of the terminal oxidase, cytochrome ba3 . We describe a purification procedure for this enzyme that yields approximately 100 mg of pure protein from 2 kg of wet mass of cells grown in < or = 50 microM O2 . Examination of the protein by SDS-polyacrylamide gel electrophoresis followed by staining with Coomassie Blue reveals one strongly staining band at approximately 35 kDa and one very weakly staining band at approximately 18 kDa as reported earlier (Zimmermann, B.H., Nitsche, C.I., Fee, J . A., Rusnak, F., and Munck, E . (1988) Proc . Natl . Acad . Sci . U.S.A . 85, 5779-5783) . By contrast, treatment of the gels with AgNO3 reveals that the larger polypeptide stains quite weakly while the smaller polypeptide stains very strongly . These results suggested the presence of two polypeptides in this protein . Using partial amino acid sequences from both proteins to obtain DNA sequence information, we isolated and sequenced a portion of the Thermus chromosome containing the genes encoding the larger protein, subunit I (cbaA), and the smaller protein, subunit II (cbaB) . The two polypeptides were isolated using reversed phase liquid chromatography, and their mole percent amino acid compositions are consistent with the proposed translation of their respective genes . The two genes appear to be part of a larger operon, but we have not extended the sequencing to identify initiation and termination sequences . The deduced amino acid sequence of subunit I includes the six canonical histidine residues involved in binding the low spin heme B and the binuclear center Cu(B)/heme A . These and other conserved amino acids are placed along the polypeptide among alternating hydrophobic and hydrophilic segments in a pattern that shows clear homology to other members of the heme- and copper-requiring terminal oxidases . The deduced amino acid sequence of the subunit II contains the CuA binding motif, including two cysteines, two histidines, and a methionine, but, in contrast to most other subunits II, it has only one region of hydrophobic sequence near its N terminus . Alignment of these two polypeptides with other cytochrome c and quinol oxidases, combined with secondary structure analysis and previous spectral studies, clearly establish cytochrome ba3 as a bona fide member of the superfamily of heme- and copper-requiring oxidases . The alignments further indicate that cytochrome ba3 is phylogenetically distant from other cytochrome c and quinol oxidases, and they substantially decrease the number of conserved amino acid residues. Mol Cell Biol, 1995 Sep, 15(9), 5173 - 9 Gene-specific signal transduction between microtubules and tubulin genes in Tetrahymena thermophila; Gu L et al.; Mammalian cells regulate tubulin mRNA abundance by a posttranscriptional mechanism dependent on the concentration of tubulin monomer . Treatment of mammalian cells with microtubule-depolymerizing drugs and microtubule-polymerizing drugs causes decreases and increases in tubulin mRNA, respectively (D . W . Cleveland, Curr . Opin . Cell Biol . 1:10-14, 1989) . In striking contrast to the case with mammalian cells, perturbation of microtubules in Tetrahymena thermophila by microtubule-depolymerizing or -polymerizing drugs increases the level of the single alpha-tubulin gene message by increasing transcription (L . A . Stargell, D . P . Heruth, J . Gaertig, and M . A . Gorovsky, Mol . Cell . Biol . 12:1443-1450, 1992) . In this report we show that antimicrotubule drugs preferentially induce the expression of one of two beta-tubulin genes (BTU1) in T . thermophila . In contrast, deciliation induces expression of both beta-tubulin genes . Tubulin gene expression was examined in a mutant strain created by transformation with an in vitro-mutagenized beta-tubulin gene that conferred resistance to microtubule-depolymerizing drugs and sensitivity to the polymerizing drug taxol and in a strain containing a nitrosoguanidine-induced mutation in the single alpha-tubulin gene that conferred the same pattern of drug sensitivities . In both cases the levels of tubulin mRNA expression from the drug-inducible BTU1 gene in the mutant cells paralleled the altered growth sensitivities to microtubule drugs . These studies demonstrate that T . thermophila has distinct, gene-specific mechanisms for modulating tubulin gene expression depending on whether ciliary or cytoplasmic microtubules are involved . They also show that the cytoplasmic microtubule cytoskeleton itself participates in a signal transduction pathway that regulates specific tubulin gene transcription in T . thermophila. J Appl Bacteriol, 1995 Sep, 79(3), 302 - 7 Amino acid requirements and peptidase activities of Streptococcus salivarius subsp . thermophilus; Neviani E et al.; The aim of this work was to investigate the relationship between amino acid requirements and peptidase enzyme systems in three Streptococcus salivarius subsp . thermophilus strains . A synthetic medium without nitrogen components and a milk (RD milk) without its non-protein nitrogen fraction were prepared with different mixtures of amino acids . The strains showed different amino acid requirements . Some amino acids proved to be essential, some were required, while others did not affect growth . In the synthetic medium, only leucine and glutamic acid were essential for growth . In RD milk, the amino acid requirements were found to be lower, with only the absence of glutamic acid causing complete inhibition of growth . Relationships between aminopeptidase activities of the strains and their amino acid requirements were observed . Strains with higher amino acid requirements were also found to express a wider range of peptidases. J Eukaryot Microbiol, 1995 Sep-Oct, 42(5), 510 - 5 A simple, efficient technique for freezing Tetrahymena thermophila; Cassidy-Hanley D et al.; We have developed a simple, efficient procedure for the long term freezing of Tetrahymena thermophila in liquid nitrogen . This technique yields excellent recovery of viable cells with all strains tested and does not require the use of a controlled rate low temperature freezer . To optimize the freezing technique, we have examined the effects of varying a number of parameters, including the physiological state of the cells prior to freezing, the time of exposure to cryoprotectant, and the rate of freezing and thawing . The frequency of viable cell recovery following freezing using this technique has been tested for a variety of different cell lines. Enzyme Microb Technol, 1995 Sep, 17(9), 816 - 25 Purification and partial characterization of a novel thermophilic carboxylesterase with high mesophilic specific activity; Wood AN et al.; An esterase activity obtained from a strain of Bacillus stearothermophilus was purified 5,133-fold to electrophoretic homogeneity with 26% recovery . The purified esterase had a specific activity of 2,032 mumol min-1 mg-1 based on the hydrolysis of p-nitrophenyl caproate at pH 7.0 and 30 degrees C . The apparent molecular mass was 50,000 +/- 2,000 daltons from sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 45,000 +/- 3,000 daltons from gel filtration . Native polyacrylamide gels stained for esterase activity showed three bands . The isoelectric points were estimated to be 5.7, 5.8, and 6.0 . Forty amino acid residues were sequenced at the N-terminus . The sequence showed no degeneracy, suggesting that the three esterases are functionally identical carboxylesterases differing by a limited number of amino acids . The enzyme showed maximum activity at pH 7.0 and was very stable at pH 6.0-8.9 with optimum stability at pH 6.0 . At this pH and 60 degrees C the half-life was 170 h . Esterase activity was totally inhibited by phenylmethanesulfonyl fluoride, parahydroxymercuribenzoate, eserine, and tosyl-L-phenylalanine, but not by ethylendiaminetetra acetic acid . The esterase obeyed Michaelis-Menten kinetics in the hydrolysis of p-nitrophenyl esters, but both Vmax and KM were protein concentration-dependent . The esterase was able to hydrolyse a number of p-nitrophenyl derivatives (amino acid derivatives and aliphatic acids with different chain lengths). EMBO J, 1995 Sep 1, 14(17), 4156 - 67 Crystal structure of glycyl-tRNA synthetase from Thermus thermophilus; Logan DT et al.; The sequence and crystal structure at 2.75 A resolution of the homodimeric glycyl-tRNA synthetase from Thermus thermophilus, the first representative of the last unknown class II synthetase subgroup, have been determined . The three class II synthetase sequence motifs are present but the structure was essential for identification of motif 1, which does not possess the proline previously believed to be an essential class II invariant . Nevertheless, crucial contacts with the active site of the other monomer involving motif 1 are conserved and a more comprehensive description of class II now becomes possible . Each monomer consists of an active site strongly resembling that of the aspartyl and seryl enzymes, a C-terminal anticodon recognition domain of 100 residues and a third domain unusually inserted between motifs 1 and 2 almost certainly interacting with the acceptor arm of tRNA(Gly) . The C-terminal domain has a novel five-stranded parallel-antiparallel beta-sheet structure with three surrounding helices . The active site residues most probably responsible for substrate recognition, in particular in the Gly binding pocket, can be identified by inference from aspartyl-tRNA synthetase due to the conserved nature of the class II active site. EMBO J, 1995 Sep 1, 14(17), 4143 - 55 Crystal structure of histidyl-tRNA synthetase from Escherichia coli complexed with histidyl-adenylate; Arnez JG et al.; The crystal structure at 2.6 A of the histidyl-tRNA synthetase from Escherichia coli complexed with histidyl-adenylate has been determined . The enzyme is a homodimer with a molecular weight of 94 kDa and belongs to the class II of aminoacyl-tRNA synthetases (aaRS) . The asymmetric unit is composed of two homodimers . Each monomer consists of two domains . The N-terminal catalytic core domain contains a six-stranded antiparallel beta-sheet sitting on two alpha-helices, which can be superposed with the catalytic domains of yeast AspRS, and GlyRS and SerRS from Thermus thermophilus with a root-mean-square difference on the C alpha atoms of 1.7-1.9 A . The active sites of all four monomers are occupied by histidyl-adenylate, which apparently forms during crystallization . The 100 residue C-terminal alpha/beta domain resembles half of a beta-barrel, and provides an independent domain oriented to contact the anticodon stem and part of the anticodon loop of tRNA(His) . The modular domain organization of histidyl-tRNA synthetase reiterates a repeated theme in aaRS, and its structure should provide insight into the ability of certain aaRS to aminoacylate minihelices and other non-tRNA molecules. Arch Microbiol, 1995 Sep, 164(3), 159 - 64 Thermococcus peptonophilus sp . nov., a fast-growing, extremely thermophilic archaebacterium isolated from deep-sea hydrothermal vents; Gonzalez JM et al.; Two extremely thermophilic archaebacteria, strains OG-1 and SM-2, were isolated from newly discovered deep-sea hydrothermal vent areas in the western Pacific ocean . These strains were cocci, obligately anaerobic Archaea about 0.7-2 microm in diameter . Optimum growth conditions for OG-1 and SM-2 were at 85-90 degrees C (range 60-100 degrees C), pH 6 (range pH 4-8), a NaCl concentration of 3% (range 1-5%), and a nutrient concentration (tryptone plus yeast extract) of 0.2% (range 0.005-5%) . Elemental sulfur stimulated the growth rate fourfold . Ammonium slightly stimulated growth . Both tryptone and yeast extract allowed growth as sole carbon sources; these isolates were not able to utilize or grow exclusively on sucrose, glucose, maltose, succinate, pyruvate, propionate, acetate, or free amino acids . OG-1 showed the fastest growth rate within the genus Thermococcus . Growth was inhibited by rifampicin . The DNA G+C content was 52 mol% . Sequencing of their 16S rDNA gene fragment indicated that these isolates belonged to the genus Thermococcus . OG-1 and SM-2 were different than the described Thermococcus species . We propose that OG-1 belongs to a new species: Thermococcus peptonophilus. Microbiology, 1995 Sep, 141 ( Pt 9), 2033 - 40 alpha-D-glucuronidases from the xylanolytic thermophiles Clostridium stercorarium and Thermoanaerobacterium saccharolyticum; Bronnenmeier K et al.; alpha-D-Glucuronidases were purified from the xylanolytic thermophiles Clostridium stercorarium and Thermoanaerobacterium saccharolyticum . This enzyme activity was found to be intracellular in each organism, with T . saccharolyticum producing much greater total activity . The specific activities of the purified enzymes (10 U mg-1 T . saccharolyticum; 1.7 U mg-1 C . stercorarium) differed by a factor of approximately 5 . For the determination of enzyme activities, 4-O-methyl-alpha-D-glucuronosyl-xylotriose was used as a substrate and the glucuronic acid released by alpha-D-glucuronidase action was quantified by a colorimetric procedure . 4-O-Methyl-alpha-D-glucuronosyl-xylotriose was the hydrolysis product that accumulated after exhaustive degradation of 4-O-methyl-alpha-D-glucuronoxylan with xylanases of C . stercorarium . Hydrolysis of side chains in high-molecular-mass glucuronoxylan could not be detected . Neither of the enzymes was able to hydrolyse the chromogenic aryl-substrate p-nitrophenyl-alpha-D-glucuronoside . Both alpha-D-glucuronidases have a dimeric structure, with monomeric molecular masses of 72 and 76 kDa for C . stercorarium and of 71 kDa for T . saccharolyticum . The pI was estimated to be 4.3 for each enzyme . While both enzymes exhibited a similar pH optimum (pH 5.5-6.5) they differed in their thermostabilities . At 60 degrees C, half-lives of 14 and 2.5 h, respectively, were determined for the alpha-D-glucuronidases of C . stercorarium and T . saccharolyticum . This description of alpha-D-glucuronidase activity in thermophilic anaerobic bacteria extends our knowledge of these enzymes, previously purified and characterized only in fungi. Gene, 1995 Aug 8, 161(1), 1 - 6 Cloning and sequence analysis of the DNA ligase-encoding gene of Rhodothermus marinus, and overproduction, purification and characterization of two thermophilic DNA ligases; Thorbjarnardottir SH et al.; In this paper we describe the cloning and sequence analysis of a gene encoding DNA ligase (Lig; EC 6.5.1.2) from the thermophilic bacterium Rhodothermus marinus (Rm) . We also describe the overexpression of the Lig-encoding genes of Rm and the thermophile, Thermus scotoductus (Ts), in Escherichia coli, and the purification and characterization of the overproduced Lig . The Rm lig gene encodes a protein of 712 amino acids (aa) with a calculated molecular mass of 79,487 Da . Comparison with published sequences of bacterial Lig revealed significant homology between the NAD(+)-utilizing Lig, and alignment of their aa sequences revealed several blocks of conserved residues . Both of the purified Lig exhibit nick-closing activity over a wide range of temperatures . Under our assay conditions the Rm Lig was active at 5-75 degrees C with apparent optimal activity above 55 degrees C . The Ts enzyme showed activity at 15-75 degrees C with optimal activity above 65 degrees C . The half-life of the Lig at 91 degrees C was estimated to be 7 min for the Rm Lig and 26 min for the Ts Lig. Biochemistry, 1995 Aug 8, 34(31), 10063 - 77 Gene cloning, expression, and characterization of the Sac7 proteins from the hyperthermophile Sulfolobus acidocaldarius; McAfee JG et al.; The genes for two Sac7 DNA-binding proteins, Sac7d and Sac7e, from the extremely thermophilic archaeon Sulfolobus acidocaldarius have been cloned into Escherichia coli and sequenced . The sac7d and sac7e open reading frames encode 66 amino acid (7608 Da) and 65 amino acid (7469 Da) proteins, respectively . Southern blots indicate that these are the only two Sac7 protein genes in S . acidocaldarius, each present as a single copy . Sac7a, b, and c proteins appear to be carboxy-terminal modified Sac7d species . The transcription initiation and termination regions of the sac7d and sac7e genes have been identified along with the promoter elements . Potential ribosome binding sites have been identified downstream of the initiator codons . The sac7d gene has been expressed in E . coli, and various physical properties of the recombinant protein have been compared with those of native Sac7 . The UV absorbance spectra and extinction coefficients, the fluorescence excitation and emission spectra, the circular dichroism, and the two-dimensional double-quantum filtered 1H NMR spectra of the native and recombinant species are essentially identical, indicating essentially identical local and global folds . The recombinant and native proteins bind and stabilize double-stranded DNA with a site size of 3.5 base pairs and an intrinsic binding constant of 2 x 10(7) M-1 for poly{dGdC}.poly{dGdC} in 0.01 M KH2PO4 at pH 7.0 . The availability of the recombinant protein permits a direct comparison of the thermal stabilities of the methylated and unmethylated forms of the protein . Differential scanning calorimetry demonstrates that the native protein is extremely thermostable and unfolds reversibly at pH 6.0 with a Tm of approximately 100 degrees C, while the recombinant protein unfolds at 92.7 degrees C. FEBS Lett, 1995 Aug 7, 369(2-3), 229 - 32 Ribosomal protein L22 from Thermus thermophilus: sequencing, overexpression and crystallisation; Davydova NL et al.; The gene for the ribosomal protein L22 from Thermus thermophilus has been sequenced and overexpressed in Escherichia coli . A multiple sequence alignment was carried out for all proteins of the L22 family reported so far . The recombinant protein was purified and crystallized . The crystals belong to the space group P2(1)2(1)2(1), with cell parameters of a = 32.6 A, b = 66.0 A, c = 67.8 A. FEBS Lett, 1995 Aug 7, 369(2-3), 158 - 60 In vivo assembly of plasmid-expressed ribosomal protein S7 of Thermus thermophilus into Escherichia coli ribosomes and conditions of its overexpression; Karginov AV et al.; Researchers still have great difficulty in isolating individual ribosomal proteins from the ribosome in quantities high enough for structural research . To this end, when studying protein S7, we created an E . coli overproducer of the recombinant protein S7 of Thermus thermophilus . The vector for expression was pQE-32 having a strong promoter of E . coli phage T5 and six triplets of His at the 5'-end . This N-terminal six His tag of the fusion protein is responsible for binding to Ni-NTA-resin and allows purifying the protein in one step . The yield of the recombinant protein was 20% and more of the total cellular proteins . In addition we have shown that the recombinant thermophilic protein is incorporated in vivo into the ribosome of E . coli despite the fact that these proteins (thermophilic and mesophilic) have a rather low homology, only 52% . This fact provides a base for the system to study functions of individual proteins. J Dairy Sci, 1995 Aug, 78(8), 1657 - 64 Effect of milks inoculated with Lactobacillus acidophilus or a yogurt starter culture in lactose-maldigesting children; Montes RG et al.; Dairy products containing live bacteria that possess lactase activity are used for dietary management of lactose maldigestion . The efficacy of acidophilus milk and the effect of consuming unfermented milk that had been inoculated with yogurt bacteria have not been examined in children . We compared scores for breath H2 excretion and symptoms of 20 lactose-maldigesting children following ingestion of 250 ml of uninoculated milk with two identical milks inoculated with 10(10) cells of Lactobacillus acidophilus or with a commercial yogurt starter culture containing 10(8) cells of Lactobacillus lactis and 10(10) cells of Streptococcus thermophilus . Nine of 10 subjects who were symptomatic following ingestion of uninoculated milk experienced a reduction in symptoms following ingestion of milk inoculated with L . acidophilus, without a decline in H2 excretion . Five of 6 subjects who were symptomatic following uninoculated milk had decreased symptoms and a significant reduction in H2 excretion following milk inoculated with the yogurt culture . For lactose-maldigesting children, milks inoculated with L . acidophilus or with a yogurt culture were associated with decreased symptoms compared with those with uninoculated milk. Protein Eng, 1995 Aug, 8(8), 763 - 7 The crystal structure of thermostable mutants of chimeric 3-isopropylmalate dehydrogenase, 2T2M6T; Sakurai M et al.; A chimeric 3-isopropylmalate dehydrogenase (IPMDH), 2T2M6T, was produced by replacing the amino acid sequences of the Thermus thermophilus enzyme with those of the Bacillus subtilis enzyme from residues 75 to 113 . Decreased thermostability of the chimeric enzyme was recovered by either evolutionary engineering (I93L) or site-directed mutagenesis (S82R) . The 3-D structures of the mutants have been determined by X-ray diffraction at 2.1 A resolution . Although S82R was refined routinely, I93L required the preliminary rigid-body refinement of each domain . The R-factors were reduced to 0.18 for both mutants . Removal of the unfavorable torsion angle at isoleucine 93 may have made I93L more thermostable than 2T2M6T . In the case of S82R, the replaced arginine residue contributed to the extra hydrogen bond with water molecules . The large replaced residue decreased the entropy of the solvent, which may have caused the improvement in enzyme thermostability . Denaturation by heating may be interpreted from these structural results. Arch Microbiol, 1995 Aug, 164(2), 91 - 7 Thermotoga subterranea sp . nov., a new thermophilic bacterium isolated from a continental oil reservoir; Jeanthon C et al.; A thermophilic, strictly anaerobic bacterium, designated strain SL1, was isolated from a deep, continental oil reservoir in the East Paris Basin (France) . This organism grew between 50 and 75 degrees C, with an optimum at 70 degrees C . It was inhibited by elemental sulfur and was able to reduce cystine and thiosulfate to hydrogen sulfide . The G+C content (40 mol%), the presence of a lipid structure unique to the genus Thermotoga, and the 16S rRNA sequence of strain SL1 indicated that the isolate belongs to the genus Thermotoga . Based on DNA-DNA hybridization, isolate SL1 does not show species-level similarity with the recognized species T . maritima, T . neapolitana, and T . thermarum . Based on this description of strain SL1, we propose the recognition of a new species: Thermotoga subterranea. J Biochem (Tokyo), 1995 Aug, 118(2), 347 - 54 Molecular cloning, expression, and characterization of chaperonin-60 and chaperonin-10 from a thermophilic bacterium, Thermus thermophilus HB8; Amada K et al.; The gene coding a chaperonin from a thermophilic bacterium, Thermus thermophilus HB8, was cloned and sequenced . The operon structure was the same as those of other bacterial chaperonins and the deduced amino acid sequences of both subunits were highly homologous to those of other chaperonins . The cloned genes of chaperonin subunits, chaperonin-10 (T.th cpn10) and chaperonin-60 (T.th cpn60), were separately expressed in Escherichia coli cells . The expressed subunits were easily purified from other host proteins including GroE, a chaperonin of E . coli . T.th cpn60 was expressed as a tetradecameric form, like GroEL of E . coli . Since chaperonin from T . thermophilus HB8 is purified as a holochaperonin, a complex of tetradecameric T.th cpn60 and heptameric T.th cpn10, a tetradecamer of T.th cpn60 without T.th cpn10 has not been obtained before . T.th cpn60 tetradecamer tended to dissociate into monomers during storage . T.th cpn10 expressed in E . coli was purified as a stable oligomer, most likely a heptamer . The activity as holo-chaperonin was reconstituted by mixing both subunits . T.th cpn60 tetradecamer itself arrested refolding of other proteins . The monomerized T.th cpn60 was easily purified from T.th cpn60 oligomer by gel permeation chromatography . Thus-obtained T.th cpn60 monomer had an ATP-independent chaperone activity, as shown for T.th cpn60 monomer isolated from authentic holo-chaperonin. J Biochem (Tokyo), 1995 Aug, 118(2), 319 - 24 Cloning and sequence analysis of the gene for phosphoenolpyruvate carboxylase from an extreme thermophile, Thermus sp; Nakamura T et al.; The ppc gene, which encodes phosphoenolpyruvate carboxylase (PEPC) of an extreme thermophile, Thermus sp., was cloned and sequenced . The ppc gene had a high G+C content (69.2%) . An open reading frame for a 857-amino-acid polypeptide was found in the gene . The calculated molecular mass was 95,632 . The amino acid sequence of Thermus PEPC was 31-37% identical and 52-57% similar to those of 17 PEPCs from mesophilic organisms . No Cys residue was found in the polypeptide, demonstrating that this residue is not essential for the catalytic activity of PEPC . The cloned gene was expressed in Escherichia coli and thermostable PEPC was obtained. Protein Sci, 1995 Aug, 4(8), 1516 - 27 The optimization of protein-solvent interactions: thermostability and the role of hydrophobic and electrostatic interactions; Spassov VZ et al.; Protein-solvent interactions were analyzed using an optimization parameter based on the ratio of the solvent-accessible area in the native and the unfolded protein structure . The calculations were performed for a set of 183 nonhomologous proteins with known three-dimensional structure available in the Protein Data Bank . The dependence of the total solvent-accessible surface area on the protein molecular mass was analyzed . It was shown that there is no difference between the monomeric and oligomeric proteins with respect to the solvent-accessible area . The results also suggested that for proteins with molecular mass above some critical mass, which is about 28 kDa, a formation of domain structure or subunit aggregation into oligomers is preferred rather than a further enlargement of a single domain structure . An analysis of the optimization of both protein-solvent and charge-charge interactions was performed for 14 proteins from thermophilic organisms . The comparison of the optimization parameters calculated for proteins from thermophiles and mesophiles showed that the former are generally characterized by a high degree of optimization of the hydrophobic interactions or, in cases where the optimization of the hydrophobic interactions is not sufficiently high, by highly optimized charge-charge interactions. Eur J Biochem, 1995 Aug 1, 231(3), 773 - 8 Coenzyme F420-dependent N5,N10-methylenetetrahydromethanopterin reductase (Mer) from Methanobacterium thermoautotrophicum strain Marburg . Cloning, sequencing, transcriptional analysis, and functional expression in Escherichia coli of the mer gene; Vaupel M et al.; The gene encoding the F420-dependent N5,N10-methylenetetrahydromethanopterin reductase (Mer), which catalyzes an intermediate step in methanogensis, was cloned and sequenced from the thermophilic Methanobacterium thermoautotrophicum strain Marburg . The gene was identified on a 3.8-kbp BamHI fragment of M . thermoautotrophicum genomic DNA using a homologous probe . The mer gene encoded an acidic protein of 321 amino acids, corresponding to a calculated molecular mass of 33,492 Da . Sequence analysis revealed the presence of a ribosome binding site, a putative promoter, and a possible terminator structure . The size of the mer mRNA was estimated as 1 kb indicating monocistronic transcription . The mer gene was expressed in Escherichia coli yielding an active enzyme of 36 kDa consistent with the apparent molecular mass described for the enzyme from M . thermoautotrophicum . Sequence comparisons revealed similarities between the F420-dependent N5,N10-methylenetetrahydromethanopterin reductase and a F420-dependent reductase involved in lincomycin biosynthesis in Streptomyces lincolnensis. J Bacteriol, 1995 Aug, 177(15), 4540 - 3 Molecular cloning of the pyrE gene from the extreme thermophile Thermus flavus; Vonstein V et al.; Mutants of the extreme thermophile Thermus flavus in the pyrimidine biosynthetic pathway (Pyr-) were isolated by resistance to 5-fluoroorotic acid . The pyrE gene, which codes for the orotate phosphoribosyltransferase, was cloned by recombination with one of the isolated Pyr- T . flavus mutant strains . It was subcloned by complementation of an Escherichia coli pyrE mutant strain and was sequenced . The deduced polypeptide sequence extends over 183 amino acids . Several independent Pyr- mutations were mapped within the pyrE locus by recombination with fragments of the cloned gene. J Bacteriol, 1995 Aug, 177(15), 4417 - 26 A multicopy plasmid of the extremely thermophilic archaeon Sulfolobus effects its transfer to recipients by mating; Schleper C et al.; A plasmid of 45 kb, designated pNOB8, was found in high copy number in a new heterotrophic Sulfolobus isolate, NOB8H2, from Japan . Dissemination of the plasmid occurred in six cultures of nine different Sulfolobus strains when small amounts of the donor were added . These mixed cultures exhibited a high average copy number of the plasmid, between 20 and 40 per chromosome, and showed a marked growth retardation . Horizontal transfer of pNOB8 was proved by isolating transcipients from mating mixtures via single colonies . In these isolates, the copy number of the plasmid appeared to be subject to a control mechanism . Cell-free filtrates of donor cultures did not transmit the plasmid, and plating of the donor on lawns of recipients did not result in plaque formation, suggesting that the transfer was not mediated by a virus . Rapid formation of cell-to-cell contacts between differently stained donor and recipient partners was demonstrated after the two strains were mixed . Electron microscopic analysis of mating mixtures revealed many cell aggregates made up of 2 to 30 cells and intercellular cytoplasmic bridges connecting two or more cells . Cells that had been transformed with purified plasmid DNA as well as transcipients isolated from mating mixtures were shown to serve as donors for further transmission of pNOB8 . The plasmid undergoes extensive genetic variations, since deletions and insertions were frequently observed in plasmid preparations from the donor strain and from mating mixtures. Curr Opin Biotechnol, 1995 Aug, 6(4), 370 - 4 Engineering thermostability: lessons from thermophilic proteins; Russell RJ et al.; As several groups begin to tap the rich pickings found in the Archaea--a vast kingdom that stretches the concept of life as we know it--the structures of proteins from hyperthermophiles are being elucidated . Certain features are beginning to emerge, such as compactness and hydrophobic clustering, but the ability to engineer these features into temperature-intolerant proteins is still some way off. Biokhimiia, 1995 Aug, 60(8), 1318 - 25 {Site-specific endonuclease BspKT8 from the thermophilic strain KT8 of Bacillus species}; Chernov AV et al.; A site-specific endonuclease capable of recognizing the sequence 5'-AAGCTT-3' was detected and purified to homogeneity from the thermophilic strain of Bacillus species KT8 . The endonuclease has a molecular mass of 34 kDa and is found in solution in a monomeric form . The activity of BspKT8 does not depend on ATP and is not stimulated by S-adenosyl-L-methionine . The enzyme displays the highest activity with a broad range of temperatures (37 degrees-48 degrees C) . Since DNA cleavage occurs in accordance with the scheme: {formula: see text} the enzyme can be assigned to the class-II of restriction endonucleases and represents an isoschizomer of HindIII. Biosci Biotechnol Biochem, 1995 Aug, 59(8), 1438 - 43 Expression of aqualysin I (a thermophilic protease) in soluble form in Escherichia coli under a bacteriophage T7 promoter; Sakamoto S et al.; The thermophilic protease aqualysin I (AQI) gene (aquI), derived from Thermus aquaticus YT-1, was inserted under the control of the bacteriophage T7 promoter in an expression plasmid . The plasmid was introduced into two strains of E . coli JM109 (DE3), one carrying and one lacking an F' episome, which carries the lacIq gene . Upon cultivation the strain carrying an F' episome produced AQI as an insoluble fusion protein (74kDa) with the T7 gene 10 protein . This insoluble protein could not be processed into mature AQI by heat treatment and thus it had no proteolytic activity . On the other hand, when the strain lacking an F' episome was used as a host cell for aquI expression, non-induced, or leaky, expression occurred, and AQI was produced in a soluble form . This soluble protein could be processed into active AQI by heat treatment . Moreover, when a low concentration of IPTG (0.0125 mM) was added, the amount of active AQI was 2.7 times greater than that produced in a batch culture without induction. Appl Microbiol Biotechnol, 1995 Aug-Sep, 43(4), 667 - 74 Direct isolation of functional genes encoding cellulases from the microbial consortia in a thermophilic, anaerobic digester maintained on lignocellulose; Healy FG et al.; Gene libraries ("zoolibraries") were constructed in Escherichia coli using DNA isolated from the mixed liquor of thermophilic, anaerobic digesters, which were in continuous operation with lignocellulosic feedstocks for over 10 years . Clones expressing cellulase and xylosidase were readily recovered from these libraries . Four clones that hydrolyzed carboxymethylcellulose and methylumbelliferyl-beta-D-cellobiopyranoside were characterized . All four cellulases exhibited temperature optima (60-65 degrees C) and pH optima (pH 6-7) in accordance with conditions of the enrichment . The DNA sequence of the insert in one clone (plasmid pFGH1) was determined . This plasmid encoded an endoglucanase (celA) and part of a putative beta-glucosidase (celB), both of which were distinctly different from all previously reported homologues . CelA protein shared limited homology with members of the A3 subfamily of cellulases, being similar to endoglucanase C from Clostridium thermocellum (40% identity) . The N-terminal part of CelB protein was most similar to beta-glucosidase from Pseudomonas fluorescens subsp . cellulosa (28% homology) . The use of zoolibraries constructed from natural or laboratory enrichment cultures offers the potential to discover many new enzymes for biotechnological applications. RNA, 1995 Aug, 1(6), 598 - 609 Identification of ribozymes within a ribozyme library that efficiently cleave a long substrate RNA; Campbell TB et al.; Positions 2-6 of the substrate-binding internal guide sequence (IGS) of the L-21 Sca I form of the Tetrahymena thermophila intron were mutagenized to produce a GN5 IGS library . Ribozymes within the GN5 library capable of efficient cleavage of an 818-nt human immunodeficiency virus type 1 vif-vpr RNA, at 37 degrees C, were identified by ribozyme-catalyzed guanosine addition to the 3' cleavage product . Three ribozymes (IGS = GGGGCU, GGCUCC, and GUGGCU) within the GN5 library that actively cleaved the long substrate were characterized kinetically and compared to the wild-type ribozyme (GGAGGG) and two control ribozymes (GGAGUC and GGAGAU) . The two control ribozymes have specific sites within the long substrate, but were not identified during screening of the library . Under single-turnover conditions, ribozymes GGGGCU, GGCUCC, and GUGGCU cleaved the 818-nt substrate 4- to 200-fold faster than control ribozymes . Short cognate substrates, which should be structureless and therefore accessible to ribozyme binding, were cleaved at similar rates by all ribozymes except GGGGCU, which showed a fourfold rate enhancement . The rate of cleavage of long relative to short substrate under single-turnover conditions suggests that GGCUCC and GUGGCU were identified because of accessibility to their specific cleavage sites within the long substrate (substrate-specific effects), whereas GGGGCU was identified because of an enhanced rate of substrate binding despite a less accessible site in the long substrate . Even though screening was performed with 100-fold excess substrate (relative to total ribozyme), the rate of multiple-turnover catalysis did not contribute to identification of trans-cleaving ribozymes in the GN5 library. Proteins, 1995 Aug, 22(4), 426 - 8 Crystallization of Thermus thermophilus histidyl-tRNA synthetase and its complex with tRNAHis; Yaremchuk AD et al.; Histidyl-tRNA synthetase (HisRS) has been purified from the extreme thermophile Thermus thermophilus . The protein has been crystallized separately with histidine and with its cognate tRNAHis . Both crystals have been obtained using the vapor diffusion method with ammonium sulphate as precipitant . The crystals of HisRS with histidine belong to the spacegroup P2(1)2(1)2 with cell parameters a = 171.3 A, b = 214.7 A, c = 49.3 A, alpha = beta = gamma = 90 degrees . A complete data set to a resolution of 2.7A with an Rmerge on intensities of 4.1% has been collected on a single frozen crystal . A partial data set collected on a crystal of HisRS in complex with tRNAHis shows that the crystals are tetragonal with cell parameters a = b = 232 A, c = 559 A, alpha = beta = gamma = 90 degrees and diffract to about 4.5 A resolution. Mol Gen Genet, 1995 Jul 28, 248(2), 236 - 41 Regulation of glycerol and maltose uptake by the IIAGlc-like domain of IINag of the phosphotransferase system in Salmonella typhimurium LT2; van der Vlag J et al.; In Enterobacteriaceae the nonphosphorylated form of IIAGlc of the phosphoenolpyruvate-dependent phosphotransferase system (PTS) can inhibit the uptake and subsequent metabolism of glycerol and maltose by binding to, and inhibiting, glycerol kinase and the MalK protein of the maltose transport system, respectively . In this report we show that the IIAGlc-like domain of the membrane-bound IIN-acetylglucosamine (IINag) of the PTS can replace IIAGlc in a Salmonella typhimurium crr mutant strain that lacks all soluble IIAGlc . The inhibition was most severe in cells which were partially induced for the glycerol or maltose uptake systems . The Streptococcus thermophilus lactose transporter LacS, which also contains a IIAGlc-like domain, could not replace IIAGlc . Neither IINag nor LacS could replace IIAGlc in activation of adenylate cyclase. Nature, 1995 Jul 27, 376(6538), 344 - 8 Neural regulation of thermotaxis in Caenorhabditis elegans; Mori I et al.; Thermal stimulus is an important environmental factor influencing animal behaviour . However, the mechanisms underlying thermosensation and thermal adaptation are poorly understood . The nematode Caenorhabditis elegans can sense a range of environmental temperatures and migrate towards the cultivation temperature on a thermal gradient . This modifiable thermotactic response provides an ideal system for studying the cellular and molecular processes involved in thermosensation and thermal information storage . We have identified neurons critical for thermotaxis by killing individual cells in live animals . The results indicate that an amphid sensory neuron, AFD, is a major thermosensory neuron . Some of the genetically defined cryophilic and thermophilic mutant phenotypes were mimicked when amphid interneurons AIY and AIZ, respectively, were killed, indicating that AIY is responsible for thermophilic movement and AIZ for cryophilic movement . We propose a neural model in which regulation of the activities of the two interneurons in opposite directions, depending on the cultivation temperature, is essential for thermotaxis. FEBS Lett, 1995 Jul 17, 368(2), 207 - 10 A minimum catalytic unit of F1-ATPase shows non-cooperative ATPase activity inherent in a single catalytic site with a Km 70 microM; Saika K et al.; F1-ATPase has three interacting catalytic sites and shows complicated kinetics . Here, we report reconstitution of a complex, most likely composed of one alpha subunit and one beta subunit, with a single catalytic site from thermophilic Bacillus PS3 F1-ATPase on the solid surface . The complex has an ATPase activity which obeys a simple non-cooperative kinetics with a Km(ATP) of 70 microM and a Vmax of 0.1 unit/mg . Different from F1-ATPase, the complex is not inactivated by 7-chrolo-4-nitrobenzofrazan . Thus, the inherent activity attributable to a single catalytic site unaffected by other catalytic sites of F1-ATPase is characterized. Biochem J, 1995 Jul 15, 309 ( Pt 2), 595 - 9 Two thermostable type II restriction endonucleases from Icelandic strains of the genus Thermus: Tsp4C I (ACN/GT), a novel type II restriction endonuclease, and Tsp8E I, an isoschizomer of the mesophilic enzyme Bgl I (GCCNNNN/NGGC); Welch SG et al.; Sixteen isolates of thermophilic bacteria from the genus Thermus, isolated from neutral and alkaline hot water springs in the southwest region of Iceland, were tested for the presence of restriction endonucleases . Extracts from five of the isolates showed evidence of the presence of restriction endonuclease activity by producing discrete nucleotide fragments when incubated at 65 degrees C with lambda phage DNA . Two of the isolates (Tsp4C and Tsp8E) were found to have particularly high levels of restriction endonuclease activity, and the respective enzymes from these two Thermus isolates were partially purified and characterized and their recognition and cleavage sites were determined . Enzyme Tsp4C I is a novel Type II restriction endonuclease recognizing the interrupted palindromic tetranucleotide sequence ACNGT, where N can be any one of the four bases in DNA . Tsp4C I, which retains full enzyme activity when incubated for 10 min at temperatures up to 76 degrees C, hydrolyses the phosphodiester bond in both strands of a double-stranded DNA substrate between the third and fourth bases of the recognition sequence (ACN/GT), generating fragments with a single base 3'-OH overhang . Enzyme Tsp8E I is a thermostable isoschizomer of the mesophilic Type II restriction endonuclease Bgl I (GCCNNNN/NGGC) {Lee, Clanton and Chirikjiam (1979) Fed . Proc . 28, 294}, generating fragments with a three base 3'-OH overhang . However, unlike Bgl I, Tsp8E I exhibits considerable thermal stability, retaining full enzyme activity when incubated for 10 min at temperatures up to 78 degrees C . Both Tsp4C I and Tsp8E I represent significant additions to the small but expanding list of the extremely thermostable restriction endonucleases. Cell, 1995 Jul 14, 82(1), 47 - 56 Linker histones are not essential and affect chromatin condensation in vivo; Shen X et al.; We have (separately) disrupted all of the expressed macronuclear copies of the HHO gene encoding macronuclear histone H1 and of the micronuclear linker histone (MLH) gene encoding the protein MicLH in Tetrahymena thermophila . These disruptions are shown to eliminate completely the expression of each protein . Strains without either linker histone grow at normal rates and reach near-normal cell densities, demonstrating that linker histones are not essential for cell survival . Histone H1 knockout (delta H1) cells have enlarged DAPI-stained macronuclei and normal-sized micronuclei, while MicLH knockout (delta MicLH) cells have enlarged micronuclei and normal-sized macronuclei . delta MicLH cells undergo mitosis normally . However, the micronuclear mitotic chromosome structure is less condensed . These studies provide evidence that linker histones are nonessential and are involved in chromatin packaging and condensation in vivo. FEBS Lett, 1995 Jul 10, 368(1), 132 - 4 Three-dimensional crystals of cytochrome-c oxidase from Thermus thermophilus diffracting to 3.8 A resolution; Soulimane T et al.; The ba3-type cytochrome-c oxidase from Thermus thermophilus has been crystallized in its native form . Crystallization was achieved by the batch and the vapour diffusion sitting drop methods using polyethylene glycol monomethyl ether 2000 as precipitating agent in the presence of octyl-beta-D-thioglucoside as detergent . The crystals diffract to 3.8 A, belong to the space group P2 or P2(1) and have unit cell dimensions of a = 80.7 A; b = 116.0 A; c = 156.9 A and beta = 104.4 degrees . The asymmetric unit contains two ba3-type oxidase molecules. FEBS Lett, 1995 Jul 10, 368(1), 117 - 21 Redox properties of the sulfhydrogenase from Pyrococcus furiosus; Arendsen AF et al.; The sulfhydrogenase from the extreme thermophile Pyrococcus furiosus has been re-investigated . The alpha beta gamma delta heterotetrameric enzyme of 153.3 kDa was found to contain 17 Fe, 17 S2-, and 0.74 Ni . The specific activity of the purified protein was 80 U/mg . Three EPR signals were found . A rhombic S = 1/2 signal (g = 2.07, 1.93, 1.89) was observed reminiscent in its shape and temperature dependence of spectra from {4Fe-4S}(2+; 1+) clusters . However, in reductive titrations the spectrum appeared at the unusually high potential Em,7.5 = -90 mV . Moreover, the signal disappeared again at Em7.5 = -328 mV . Also, two other signals appear upon reduction: a near-axial (g = 2.02, 1.95, 1.92) S = 1/2 spectrum (Em,7.5 = -303 mV) indicative for the presence of a {2Fe-2S}(2+; 1+) cluster, and a broad spectrum of unknown origin with effective g-values 2.25, 1.89 (Em,7.5 = -310 mV) . We hypothesize that the latter signal is caused by magnetic interaction of the rhombic signal and a third cluster. Biochem Biophys Res Commun, 1995 Jul 6, 212(1), 77 - 83 Multi-frequency EPR evidence for a binuclear CuA center in cytochrome c oxidase: studies with a 63Cu- and 65Cu-enriched, soluble domain of the cytochrome ba3 subunit II from Thermus thermophilus; Fee JA et al.; We have recorded multi-frequency EPR spectra of 63Cu- and 65Cu-labeled, water-soluble CuA-protein from the cytochrome ba3 of T . thermophilus . The spectrum taken at the highest frequency (34.03 GHz) shows no hyperfine structure and is nominally axial with apparent gz approximately 2.18 and gxy approximately 2.00 . The spectrum taken at the lowest frequency (3.93 GHz) shows a rich hyperfine structure . Analyses of the spectra show that the observed splitting arises from an interaction of the unpaired electron with two Cu nuclei and support the notion that CuA is a mixed-valent {Cu(II)/Cu(I)} complex in which the unpaired electronic spin is distributed evenly over the two Cu ions. Biochim Biophys Acta, 1995 Jul 3, 1250(1), 60 - 8 Steady state kinetics of the glutamate dehydrogenase from an archaebacterial extreme thermophile, isolate AN1; Hudson RC et al.; A steady state kinetic study was carried out with the glutamate dehydrogenase from the thermophilic, archaebacterial isolate AN1 . Initial velocity studies of the oxidative deamination reaction showed the mechanism is sequential and indicated that the order of substrate addition is random, while inhibition studies with products and substrate analogues suggested a strong preference for NADP+ to bind first . Initial velocity studies of the reductive amination reaction showed that the mechanism is sequential and indicated that the order of substrate addition is random, while product inhibition studies and the effect of substrate saturation on the initial velocity suggested that the preferred order of substrate addition is NADPH, 2-ketoglutarate, ammonia. Proc Natl Acad Sci U S A, 1995 Jul 3, 92(14), 6364 - 8 An activity gel assay detects a single, catalytically active histone acetyltransferase subunit in Tetrahymena macronuclei; Brownell JE et al.; Macronuclei of the ciliated protozoan Tetrahymena thermophila possess a histone acetyltransferase activity closely associated with transcription-related histone acetylation . Nothing definitive is known concerning the polypeptide composition of this activity in Tetrahymena or any comparable activity from any cellular source . An acetyltransferase activity gel assay was developed which identifies a catalytically active subunit of this enzyme in Tetrahymena . This activity gel assay detects a single polypeptide of 55 kDa (p55) in crude macronuclear extracts, as well as in column-purified fractions, which incorporates {3H}acetate from {3H}acetyl-CoA into core histone substrates polymerized directly into SDS polyacrylamide gels . p55 copurifies precisely with acetyltransferase activity through all chromatographic steps examined, including reverse-phase HPLC . Gel-filtration chromatography of this activity indicates a molecular mass of 220 kDa, suggesting that the native enzyme may consist of four identical subunits of 55 kDa . Furthermore, p55 is tightly associated with di- and greater polynucleosomes and therefore may be defined as a component of histone acetyltransferase type A--i.e., chromatin associated. Int J Syst Bacteriol, 1995 Jul, 45(3), 495 - 9 DNA relatedness of Thermus strains, description of Thermus brockianus sp . nov., and proposal to reestablish Thermus thermophilus (Oshima and Imahori) Williams RA, Smith KE, Welch SG, Micallef J, Sharp RJ. Aerobic, thermophilic, gram-negative bacteria obtained from Yellowstone National Park that were placed in the genus Thermus on the basis of phenotypic data were examined by chemotaxonomic techniques to determine their peptidoglycan compositions, their respiratory quinones, their mean DNA base compositions, and their levels of DNA-DNA homology as determined by both the filter hybridization and reassociation rate methods . These isolates from hot springs included Thermus aquaticus strains and strains of a new genospecies . We propose the name Thermus brockianus for this new genospecies; strain YS38 is the type strain of this taxon . A collection of 10 strains, including the type strain of "Thermus thermophilus", which were isolated from widely separated geothermal sites, exhibited high levels of DNA-DNA homology with each other and had similar physiological properties . Therefore, we propose that the species Thermus thermophilus (Oshima and Imahori) should be reestablished, with strain HB8 as the type strain. Int J Syst Bacteriol, 1995 Jul, 45(3), 454 - 61 Isolation and characterization of a novel alkalitolerant thermophile, Anaerobranca horikoshii gen . nov., sp . nov; Engle M et al.; Nine moderately alkalitolerant thermophilic bacteria with similar properties were isolated from water and soil samples obtained from Yellowstone National Park . These Gram-type-positive, rod-shaped bacteria produce cells with primary branches . The cells are peritrichous and exhibit only slight tumbling motility . At 60 degrees C the pH range for growth is 6.9 to 10.3, and the optimum pH is 8.5 . At pH 8.5 the temperature range for growth is 34 to 66 degrees C, with an optimum temperature of 57 degrees C . The strains are mainly proteolytic . The fermentation products from yeast extract are acetate, CO2, and H2 . Fumarate added to minimal medium containing yeast extract is stoichiometrically converted to succinate, indicating that it is used as an alternative electron acceptor . The DNA G + C content is 33 to 34 mol% . On the basis of its unique properties, such as branch formation, growth at alkaline pH values at elevated temperatures, and the relative distance of its 16S rRNA sequence from those of other known bacteria, we propose that strain JW/YL-138T (T = type strain) and eight similar strains represent a new genus and species, Anaerobranca horikoshii . Strain JW/YL-138 is designated the type strain of the type species, A . horikoshii, which was named in honor of Koki Horikoshi, a pioneer in the field of alkaliphilic bacteria. Int J Syst Bacteriol, 1995 Jul, 45(3), 441 - 8 Bacillus infernus sp . nov., an Fe(III)- and Mn(IV)-reducing anaerobe from the deep terrestrial subsurface; Boone DR et al.; Bacillus infernus sp . nov . was isolated from ca . 2,700 m below the land surface in the Taylorsville Triassic Basin in Virginia . B . infernus was a strict anaerobe that grew on formate or lactate with Fe(III), MnO2, trimethylamine oxide, or nitrate (reduced to nitrite) as an electron acceptor, and it also grew fermentatively on glucose . Type strain TH-23 and five reference strains were gram-positive rods that were thermophilic (growth occurred at 61 degrees C), halotolerant (good growth occurred in the presence of Na+ concentrations up to 0.6 M), and very slightly alkaliphilic (good growth occurred at pH 7.3 to 7.8) . A phylogenetic analysis of its 16S rRNA indicated that B . infernus should be classified as a new species of the genus Bacillus . B . infernus is the only strictly anaerobic species in the genus Bacillus. Anal Biochem, 1995 Jul 1, 228(2), 330 - 5 Purification of aminoacyl-tRNA by affinity chromatography on immobilized Thermus thermophilus EF-Tu.GTP; Ribeiro S et al.; Elongation factor Tu from Thermus thermophilus containing six histidine residues on its C-terminus, EF-Tu(CHis6), was used for purification of aminoacyl-tRNA isoacceptors, from aminoacylated bulk tRNA, by affinity chromatography . Preformed aminoacyl-tRNA.EF-Tu(CHis6).GTP ternary complexes were immobilized on Ni(2+)-nitriloacetic acid agarose and the aminoacyl-tRNA was eluted at high ionic strength or with buffers containing GDP . Compared to alternative methods, the reported immobilization by C-terminal histidine residues does not lead to loss of EF-Tu's affinity for aminoacyl-tRNA . The method is well suited for preparative isolation of aminoacylated tRNA isoacceptors and as an analytical tool to test tRNA composition and aminoacylation of tRNA in crude cellular extracts. Genetics, 1995 Jul, 140(3), 989 - 1005 Effects of nullisomic chromosome deficiencies on conjugation events in Tetrahymena thermophila: insufficiency of the parental macronucleus to direct postzygotic development; Ward JG et al.; Conjugation fails postzygotically after mating of Tetrahymena cells that have wild-type parental macronuclei but harbor noncomplementing nullisomic parental germline deficiencies . Failures begin shortly after formation of the new macronuclear precursor (anlage) and completion of the first step in elimination of the parental macronucleus (pycnosis) . Conjugants fail to complete pair separation, to eliminate one new micronucleus, and to amplify anlage DNA, and they eventually die . Some deficiencies block resorption of the pycnotic parental macronucleus, but we find no evidence for its regeneration . Some deficiencies cause aberrant anlage DNA loss . Those that do not cause DNA loss are epistatic to those that do, indicating that normal anlage development requires the dependent function of at least two types of genes . The possibility that these genes are involved in developmentally regulated anlage DNA rearrangements is discussed . Each observed conjugation defect indicates insufficiency of the parental macronucleus to direct postzygotic development and can be explained by the deficiency of essential conjugation genes that are expressed from the anlage . The failure of nullisomic conjugants to complete pair separation indicates a requirement for gene products, expressed from the early anlage or its precursors, soon after anlage first differentiate. Nat Struct Biol, 1995 Jul, 2(7), 537 - 47 Structure of phenylalanyl-tRNA synthetase from Thermus thermophilus; Mosyak L et al.; The crystal structure of phenylalanyl-tRNA synthetase from Thermus thermophilus, solved at 2.9 A resolution, displays (alpha beta)2 subunit organization . Unexpectedly, both the catalytic alpha- and the non-catalytic beta-subunits comprise the characteristic fold of the class II active-site domains . The alpha beta heterodimer contains most of the building blocks so far identified in the class II synthetases . The presence of an RNA-binding domain, similar to that of the U1A spliceosomal protein, in the beta-subunit is indicative of structural relationships among different families of RNA-binding proteins . The structure suggests a plausible catalytic mechanism which explains why the primary site of tRNA aminoacylation is different from that of the other class II enzymes. J Ind Microbiol, 1995 Jul, 15(1), 39 - 44 Expression of Streptomyces melC and choA genes by a cloned Streptococcus thermophilus promoter STP2201; Solaiman DK et al.; Streptococcus thermophilus (ST) chromosomal DNA (chr DNA) fragments having promoter activity were cloned and selected in Escherichia coli using a chloramphenicol acetyltransferase- (cat-) based promoter-probe vector pKK520-3 . Insertion of a promoterless streptomycete melanin biosynthesis operon (melC) downstream from the promoters of the library further identified clone STP2201 as a strong promoter in E . coli . Subcloning of a STP2201-melC DNA fragment into the pMEU-series S . thermophilus-E . coli shuttle vectors yielded pEU5xML2201x plasmids that conferred Mel+ phenotype to E . coli . The pEU5aML2201a was further shown to afford a high level of tyrosinase pro-anti-tyrosinase antiserum in S . thermophilus . Substituting melC with a streptomycete cholesterol oxidase gene (choA) in the same orientation yielded pEU5aCH2201a that conferred ChoA activity to an E . coli transformant at a level of (1.06 +/- 0.15) x 10(-7) units mg-1 protein . Introduction of this plasmid into S . thermophilus by electrotransformation yielded ChoA+ transformant that produced the enzyme at about 25% of the level found in E . coli. Int J Biochem Cell Biol, 1995 Jul, 27(7), 729 - 39 Characterisation of a thermostable pepstatin-insensitive acid proteinase from a Bacillus sp; Prescott M et al.; An acid proteinase, Wai 21a, produced by a thermophilic Bacillus species (strain Wai 21a) has been purified to homogeneity by cation-exchange chromatography, phenyl-Sepharose chromatography and anion-exchange chromatography . A pI of 3.8 was determined by isoelectric focussing . The protein contained some associated carbohydrate (20 mol hexose equiv/mol proteinase) . Optimal proteolytic activity was observed at pH 3.0 (at 60 degrees C) . The Leu15-Tyr16 bond was the major site of hydrolysis for the oxidized B chain of insulin . Enzyme activity was not affected by inhibitors of the cysteine, metallo or serine class of proteinases . The aspartate proteinase inhibitor, pepstatin, did not inhibit enzyme activity . Inhibition of enzyme activity by 1,2-epoxy-3-(p-nitrophenoxy)-propane indicated the presence of at least one carboxyl group essential to the catalytic mechanism of the enzyme . Proteinase activity was inhibited by diazoacetyl-DL-norleucine methyl ester in a slow and non-specific manner atypical of pepstatin-sensitive aspartate proteinases . Wai 21a proteinase may be classified as member of the pepstatin-insensitive group of aspartate proteinases . The thermal stability at pH 3.0 and 60 degrees C increased 2.1-fold (t1/2, 4.5-9.7 hr) in the presence of 5 mM Ca++ . An increase in both pH (3.0-4.5) and Ca++ concentration (0-30 mM) resulted in a 15-fold increase (t1/2, 15-230 min) in thermal stability at 75 degrees C . The amino acid composition of Wai 21a proteinase was found to be similar to other pepstatin-insensitive proteinases from bacterial sources and in particular similar to the other pepstatin-insensitive proteinases from bacterial sources and in particular similar to the thermostable enzyme, kumamolysin. Plant Mol Biol, 1995 Jul, 28(4), 759 - 66 Cloning and sequencing of the nitrate transport system from the thermophilic, filamentous cyanobacterium Phormidium laminosum: comparative analysis with the homologous system from Synechococcus sp . PCC 7942; Merchan F et al.; A genomic region from the filamentous, thermophilic non-N2-fixing cyanobacterium Phormidium laminosum was cloned and sequenced . It includes the nitrite reductase gene (nirA) and three other genes (nrtA, B and C) located downstream of nirA, which are related to the nitrate transport system on the basis of a comparison with the homologous system from Synechococcus sp . PCC 7942 . No additional nitrate assimilation-related genes were identified in about 5 kb sequenced downstream of nrtC . All four genes are arranged as an operon with a promoter-like region upstream of the nirA gene . Transcripts of these nitrate assimilation genes accumulated after long periods of nitrogen starvation . This operon also contains inverted repeat sequences in the intercistronic regions which might be involved in mRNA processing or stability. J Eukaryot Microbiol, 1995 Jul-Aug, 42(4), 422 - 7 Monoclonal antibodies reveal complex structure in the membrane skeleton of Tetrahymena; Williams NE et al.; Twelve monoclonal antibodies were raised that are specific for the membrane skeleton of Tetrahymena . Five were directed against T . pyriformis and seven were directed against T . thermophila . Some cross-reactivity between species was found . Each monoclonal antibody recognized one of the three major components of epiplasm, i.e . the bands A, B, and C identified in electrophoretic separations of epiplasmic proteins . It was found, using these antibodies, that the epiplasmic proteins A, B and C have overlapping but independent distributions within the cell. Appl Environ Microbiol, 1995 Jul, 61(7), 2798 - 801 Effect of genome size and rrn gene copy number on PCR amplification of 16S rRNA genes from a mixture of bacterial species; Farrelly V et al.; In order to assess the effect of genome size and number of 16S rRNA genes (rDNAs) on the quantities of PCR-generated partial 16S rDNA fragments, equimolar amounts of DNA from pairs of different species for which these parameters are known were subjected to gene amplification . The experimentally determined ratio of PCR products obtained, as determined by image analysis of SYBR-Green I-stained amplification products, corresponded well with the predicted ratio calculated from the number of rrn genes per equimolar amounts of DNA in mixtures of Escherichia coli and "Thermus thermophilus" and of Pseudomonas aeruginosa and "T . thermophilus." The values for the pair of Bacillus subtilis and "T . thermophilus" showed greater deviations from the predicted value . The dependence of the amount of 16S rDNA amplification product on these two parameters makes it impossible to quantify the number of species represented in 16S rDNA clone libraries of environmental samples as long as these two parameters are unknown for the species present. Appl Environ Microbiol, 1995 Jul, 61(7), 2762 - 4 Pressure stabilization is not a general property of thermophilic enzymes: the adenylate kinases of Methanococcus voltae, Methanococcus maripaludis, Methanococcus thermolithotrophicus, and Methanococcus jannaschii; Konisky J et al.; The application of 50-MPa pressure did not increase the thermostabilities of adenylate kinases purified from four related mesophilic and thermophilic marine methanogens . Thus, while it has been reported that some thermophilic enzymes are stabilized by pressure (D . J . Hei and D . S . Clark, Appl . Environ . Microbiol . 60:932-939, 1994), hyperbaric stabilization is not an intrinsic property of all enzymes from deep-sea thermophiles. Appl Environ Microbiol, 1995 Jul, 61(7), 2566 - 71 Microbial degradation and humification of the lawn care pesticide 2,4-dichlorophenoxyacetic acid during the composting of yard trimmings; Michel FC Jr et al.; The fate of the widely used lawn care herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) during the composting of yard trimmings consisting of primarily leaves and grass is an important unexplored question . In this study, we determined the extent of 2,4-D mineralization, incorporation into humic matter, volatilization, and sorption during the composting of yard trimmings . Yard trimmings (2:1 {wt/wt} leaves-grass) were amended with 14C-ring-labeled 2,4-D (17 mg/kg of dry weight) and composted in a temperature-controlled laboratory scale compost system . During composting, thermophilic microbes were numerically dominant, reaching a maximum of 2 x 10(11)/g . At the end of composting, 46% of the organic matter (OM) present in the yard trimmings was lost and the compost was stable, with an oxygen uptake rate of 0.09 mg of O2 per g of OM per h, and was well humified (humification index, 0.39) . Mineralization of the OM temporally paralleled mineralization of 2,4-D . In the final compost, 47% of the added 2,4-D carbon was mineralized, about 23% was complexed with high-molecular-weight humic acids, and about 20% was not extractable (humin fraction) . Less than 1% of the added 14C was present in water expressed from the finished compost, suggesting a low potential for leaching of 2,4-D . Very little volatilization of 2,4-D occurred during composting . It is of interest that our results indicate active mineralization of 2,4-D at composting temperatures of 60 degrees C because microbial 2,4-D degradation at thermophilic temperatures has not been previously documented. J Bacteriol, 1995 Jul, 177(13), 3668 - 72 Pressure effects on the composition and thermal behavior of lipids from the deep-sea thermophile Methanococcus jannaschii; Kaneshiro SM et al.; The deep-sea archaeon Methanococcus jannaschii was grown at 86 degrees C and under 8, 250, and 500 atm (1 atm = 101.29 kPa) of hyperbaric pressure in a high-pressure, high-temperature bioreactor . The core lipid composition of cultures grown at 250 or 500 atm, as analyzed by supercritical fluid chromatography, exhibited an increased proportion of macrocyclic archaeol and corresponding reductions in aracheol and caldarchaeol compared with the 8-atm cultures . Thermal analysis of a model core-lipid system (23% archaeol, 37% macrocyclic archaeol, and 40% caldarchaeol) using differential scanning calorimetry revealed no well-defined phase transition in the temperature range of 20 to 120 degrees C . Complementary studies of spin-labeled samples under 10 and 500 atm in a special high-pressure, high-temperature electron paramagnetic resonance spectroscopy cell supported the differential scanning calorimetry phase transition data and established that pressure has a lipid-ordering effect over the full range of M . jannaschii's growth temperatures . Specifically, pressure shifted the temperature dependence of lipid fluidity by ca . 10 degrees C/500 atm. Cell Mol Biol (Noisy-le-grand), 1995 Jul, 41(5), 625 - 38 Detection and identification of Campylobacter spp . using the polymerase chain reaction; Giesendorf BA et al.; Since Campylobacters have fastidious growth requirements and conventional detection and identification requires at least 4-6 days, the development of fast but reliable detection procedures is needed . Although methods based on DNA probe technology have been developed, these are not sensitive enough for the detection of Campylobacter spp . in food products . Therefore a PCR procedure based on the amplification of the 16S rRNA gene was developed that specifically detects the thermophilic Campylobacter species . This assay provides an excellent tool for the rapid and sensitive isolation and identification of Campylobacter spp . from chicken samples . In order to further identify the different Campylobacter spp., which are difficult to distinguish by conventional methods, PCR mediated approximately DNA typing was used to select species-specific DNA probes . This combination of PCR fingerprinting and probe hybridization results in a highly specific identification assay and provides an example of specific test development without the prior need for DNA sequence information . PCR mediated DNA typing was also used to study the epidemiology of diarrheal diseases caused by Campylobacter spp . Using primers complementary to dispersed repetitive DNA sequences and arbitrarily chosen DNA motifs PCR fingerprinting has proven to be a fast, highly discriminative and relatively simple method that can be applied in epidemiological investigations on Campylobacter infections . Besides this application of PCR fingerprinting for typing of Campylobacter spp . this method can also be used for the development of specific DNA probes. Microbiology, 1995 Jul, 141 ( Pt 7), 1731 - 8 A gene encoding a thermophilic alkaline serine proteinase from Thermus sp . strain Rt41A and its expression in Escherichia coli; Munro GK et al.; The extreme thermophile Thermus sp . strain Rt41A produces an extracellular alkaline serine proteinase during growth . This enzyme is stable for more than 24 h at 70 degrees C and has a pH optimum of 8.0 . The proteinase gene was identified using primers designed to amplify a region between two highly conserved amino acid motifs in subtilisin-like proteinases and the PCR product was used to identify a genomic fragment containing the gene . The amino acid sequence deduced from the Rt41A gene contained a region identical to that obtained by amino-terminal sequencing of purified Rt41A proteinase . Comparison of the entire derived peptide sequence with other subtilisin-like serine proteinases revealed significant homologies, especially with aqualysin I from Thermus aquaticus YT-1 and with exoprotease A from Vibrio alginolyticus . The Rt41A proteinase was expressed in Escherichia coli as a fusion protein with glutathione-S-transferase as an aid for purification and to overcome difficulties experienced with other plasmid vectors which produced inactive protein . The enzyme is inactive as synthesized and activation was shown to be temperature-dependent, with shorter incubation times required at higher temperatures; removal of the hydrophobic signal peptide from the start of the gene reduced the time required for activation to less than a third of that required if the signal peptide was present. Bioorg Khim, 1995 Jul, 21(7), 492 - 7 {Structural properties of ribosomal protein S8 from the extreme thermophile Thermus thermophilus}; Vysotskaia VS et al.; The gene of ribosomal protein S8 from the extreme thermophile Thermus thermophilus was expressed in E . coli using the strain BL21(DE3) and vector pET3-1 . A method of isolating this protein from the super producing strain was developed, which makes it possible to obtain 8-12 mg of product from 11 of culture . The secondary structure of protein S8 was determined by using CD spectroscopy . The protein was shown to be highly resistant to denaturants. Yeast, 1995 Jul, 11(9), 873 - 83 A 37.5 kb region of yeast chromosome X includes the SME1, MEF2, GSH1 and CSD3 genes, a TCP-1-related gene, an open reading frame similar to the DAL80 gene, and a tRNA(Arg); Rasmussen SW; The complete DNA sequence of cosmid clone p59 comprising 37,549 bp derived from chromosome X was determined from an ordered set of subclones . The sequence contains 14 open reading frames (ORFs) containing at least 100 consecutive sense codons . Four of the ORFs represent already known and sequenced yeast genes: B645 is identical to the SME1 gene encoding a protein kinase, required for induction of meiosis in yeast, D819 represents the MEF2 gene probably encoding a second mitochondrial elongation factor-like protein, D678 is identical to the yeast GSH1 gene encoding gamma-glutamylcysteine synthetase and B746 is identical to the CSD3 gene, which plays an as yet unidentified role in chitin biosynthesis and/or its regulation . The deduced amino acid sequence of A550 is 63% identical to the Cc eta subunit of a murine TCP-1-containing chaperonin and more than 35% identical to thermophilic factor 55 from Sulfolobus shibatae, as well as to a number of proteins belonging to the chaperonin TCP-1 family . Open reading frame F551 exhibits homology to two regions of the DAL80 gene located on yeast chromosome XI encoding a pleiotropic negative regulatory protein . In addition, extensive homology was detected in three regions including parts of ORFs A560, B746/CSD3 and the incomplete ORF C852 to three consecutive ORFs of unknown function in the middle of the right arm of chromosome XI . Finally, the sequence contained a tRNA(Arg3) (AGC) gene. Mol Biol (Mosk), 1995 Jul-Aug, 29(4), 942 - 9 {Use of thermostable DNA polymerase from Thermus thermophilus KTP in a combined reverse transcription and amplification reaction for detecting CD4 receptor mRNA}; Glukhov AI et al.; The RT/PCR method was applied to study a possible use of Tth DNA-polymerase for coupled reaction of reverse transcription and polymerase chain reaction (RT/PCR) on the CD-4 receptor mRNA template in the total cellular RNA . The conditions for detecting the CD-4 receptor mRNA were optimized . The pH-optimum for RT reaction was 8.8 . The influence of Mn2+, Cu2+, Co2+, and Cd2+ cations in RT and PCR reaction was investigated . The efficiency of the RT reaction was shown to be the highest in the presence of Mn2+ (optimal concentration 1 mM) . At Mn2+ concentration > or = 3 mM complete inhibition of RT/PCR was observed . The Tth DNA polymerase in RT/PCR was shown to be more effective than Taq DNA polymerase . The Tth DNA polymerase allows observation of the specific product in the gel containing ethidium bromide using 20 ng of the total RNA . High sensitivity and specificity of RT/PCR performed with the Tth DNA polymerase allow its wide application in the detection, quantitative analysis and cloning of cellular and viral RNAs. Mol Biol (Mosk), 1995 Jul-Aug, 29(4), 930 - 41 {Use of thermostable DNA polymerase from Thermus thermophilus KTP in a combined reverse transcription and amplification reaction of detecting interleukin 2alpha RNA and determining expression of the multidrug resistance gene (MDR-1)}; Grebennikova TV et al.; According to our data native Tth DNA polymerase displays higher reverse transcription activity than Taq DNA polymerase . This allows one to use Tth DNA polymerase in the complete reaction of reverse transcription and amplification (RT/PCR) . We used this enzyme to synthesize the interleukine (IL-2 alpha) RNA template synthesized by the RT/PCR method in vitro . The conditions for RNA IL-2 alpha detection were optimized . The maximum yield of the specific product was obtained at pH 8.5-9.0 . The influence of bivalent cations on the efficiency of RT reaction of coupled R