Biochemistry, 1996 Jul 30, 35(30), 9880 - 91 Metal binding sites of H(+)-ATPase from chloroplast and Bacillus PS3 studied by EPR and pulsed EPR spectroscopy of bound manganese(II); Buy C et al.; The metal binding sites of isolated F1 ATPase from spinach chloroplasts (CF1) and from the thermophilic bacterium Bacillus PS3 (TF1) have been studied by EPR and pulsed EPR spectroscopy using Mn(II) as a paramagnetic probe . After dialysis in the presence of EDTA, purified CF1 retains 0.14 +/- 0.07 Mg(II) and approximately 0.75 +/- 0.25 ADP . TF1 retains 0.31 +/- 0.03 Mg(II) and 0.08 +/- 0.01 nucleotide (ADP + ATP) after the same treatment . Supplementing known quantities of Mn(II) to metal-depleted CF1 allowed a spectroscopic characterization of the bound Mn(II) cations, for which the EPR spectra at X- and Q-band are reported . The zero field splitting parameters of Mn(II) are derived from the simulation of the EPR signal recorded at Q-band for a sample supplemented with 0.3 Mn/CF1 . The values, magnitude of D approximately 200 x 10(-4) cm-1 and magnitude of E approximately 40 x 10(-4) cm-1 suggest that the Mn(II) binds to CF1 in a slightly distorted environment . The ESEEM spectra of complexes of Mn(II) with CF1 were also recorded for different Mn/CF1 ratios . For a complex with 0.8 Mn/CF1, the ESEEM spectrum shows two frequencies at 3.7 and 8.6 MHz that are attributed to the magnetic coupling with 31P with a hyperfine coupling constant of magnitude of A approximately 5.3 MHz, reflecting the interaction with a phosphate group from the endogenous ADP molecule . This demonstrates close proximity of the strong affinity metal site M1 and the endogenous ADP binding site N1, and binding of the ADP beta-phosphate to the divalent metal cation . For Mn(II) complexes with higher Mn/CF1 ratios, new frequency components below approximately 5 MHz are resolved in the spectra in addition to the peaks from 31P . From a comparison of the CF1 spectra and their magnetic field dependence across the Mn(II) EPR line shape with those of Mn(II) complexes with imidazole, glycine, poly-L-lysine, and nucleotide ligands, it is concluded that additional metal binding sites are filled at higher Mn contents and that these involve 14N donors . It is suggested that the most probable set of ligands of the divalent metal(s) for these additional metal sites in CF1 includes a lysine residue, in line with a previous proposal {Houseman, A . L . P., Morgan, L., LoBrutto, R., & Frasch, W . D . (1994) Biochemistry 33, 4910-4917} . Similar experiments for a Mn(II) complex with TF1 (0.4 Mn/TF1) showed no interaction with 31P; instead modulations are detected in the ESEEM below approximately 5 MHz that are attributed to a 14N ligand . This is tentatively attributed to the deprotonated amine of Lys-162 from a beta subunit, on the basis of the structural data available for the mitochondrial F1 complex . Addition of the substrate ATP to this Mn.TF1 complex leads to the formation of a ternary Mn.TF1.ATP complex with coordination of the Mn(II) by a phosphate group from the ATP as judged from the ESEEM results (magnitude of A(31P) approximately 4.5 MHz) . An increase in the hyperfine coupling constant of 31P of the phosphate bound to Mn(II) to magnitude of A(31P) approximately 5.1 MHz is observed after incubation of the ternary complex at room temperature . This is interpreted as a significant rearrangement of the coordination sphere of the Mn(II) in the M1 site of the Mn.TF1.ATP complex and may reflect conformational changes of catalytic significance that occur in the nucleotide binding site during unisite hydrolysis of ATP to ADP by this complex. Biochemistry, 1996 Jul 30, 35(30), 9792 - 6 Stereospecificity of thermostable ornithine 5-aminotransferase for the hydrogen transfer in the L- and D-ornithine transamination; Jhee KH et al.; The thermostable ornithine 5-aminotransferase of a thermophile, Bacillus sp . YM-2, is unique in acting on both enantiomers of ornithine, although less effectively on the D-enantiomer . We studied the stereospecificity of the enzyme for the hydrogen abstraction from C-5 of the substrate moiety and the addition and removal of the hydrogen at C-4' of the cofactor (pyridoxal phosphate and pyridoxamine phosphate) moiety of the external Schiff base intermediate in the transamination of L- and D-ornithine . L- and D-{5-3H}ornithines were prepared by incubation of L- and D-ornithines with the enzyme in 3H2O, respectively . When the L-{5-3H}ornithine was incubated with L-ornithine 5-aminotransferase of a mesophile, Bacillus sphaericus, which catalyzes the stereospecific abstraction of pro-S hydrogen from C-5 of L-ornithine, most of the tritium was released into the solvent . The D-{5-3H}ornithine also reacted with the enzyme of B . sphaericus in the presence or absence of the amino acid racemase of Pseudomonas putida . Tritium was released only in the presence of the racemase, which catalyzes the racemization of ornithine but does not act on C-5 of ornithine . These results show that the Bacillus sp . YM-2 ornithine 5-aminotransferase stereospecifically abstracts the pro-S hydrogen from C-5 of L- and D-ornithine . When the apo form of the enzyme was incubated with pyridoxamine 5'-phosphate that was stereospecifically tritiated at C-4' and 2-oxoglutarate in the presence of L-ornithine or D-ornithine, tritium was released exclusively from (4'S)-{4'-3H}pyridoxamine . Therefore, addition and abstraction of hydrogen at C-4' of the cofactor moiety stereospecifically occur on the si face of the external Schiff base intermediate in the overall transamination catalyzed by Bacillus sp . YM-2 ornithine 5-aminotransferase irrespective of the C-2 configuration of the amino donor. J Biol Chem, 1996 Jul 26, 271(30), 17687 - 91 Catalytic properties of human manganese superoxide dismutase; Hsu JL et al.; The depletion of superoxide catalyzed by human manganese superoxide dismutase (MnSOD) was observed spectrophotometrically by measuring the absorbance of superoxide at 250-280 nm following pulse radiolysis and by stopped-flow spectrophotometry . Catalysis showed an initial burst of activity lasting approximately 1 ms followed by the rapid emergence of a greatly inhibited catalysis of zero-order rate . These catalytic properties of human MnSOD are qualitatively similar to those reported for MnSOD from Thermus thermophilus (Bull, C., Niederhoffer, E . C., Yoshida, T., and Fee, J . A.(1991) J . Am . Chem . Soc . 113, 4069-4076) . However, there are significant quantitative differences; the emergence of the inhibited form is approximately 30-fold more rapid for human MnSOD . The turnover number for human MnSOD at pH 9.4 and 20 degrees C was kcat = 4 x 10(4) s-1 and kcat/Km = 8 x 10(8) M-1 s-1, determined by a simulated fit of the model of Bull et al . (1991) to the pulse radiolysis data . We also report that the maximum of the visible absorption spectrum of human MnSOD (epsilon480 = 525 M-1 cm-1) showed a strong dependence on pH that could be described by an ionization of pKa 9.4 +/- 0.1 with a maximum at low pH. J Biol Chem, 1996 Jul 26, 271(30), 18128 - 33 Catalytic activities of alpha3beta3gamma complexes of F1-ATPase with 1, 2, or 3 incompetent catalytic sites; Amano T et al.; In order to know how many functional catalytic sites are necessary for ATPase activity of F1-ATPase from a thermophilic Bacillus PS3, a new method of isolating homogeneous preparations of the alpha3beta3gamma complex with 1, 2, or 3 incompetent catalytic sites was developed . Ten glutamic acids (Glu.Tag) were linked to the C terminus of the catalytically incompetent beta(E190Q) subunit . The Glu.Tag itself did not affect ATPase activity of the complexes . Two kinds of alpha3beta3gamma complexes, one containing beta(wild-type) and the other Glu.Tag-linked beta(E190Q), were mixed, urea-denatured, and dialyzed, and alpha3beta3gamma complexes were reconstituted . Each of the complexes containing a different number of Glu.Tag-linked beta(E190Q) was separated by anion-exchange chromatography and analyzed . The results were as follows . 1) Normal steady-state ATPase activity requires three intact catalytic sites . 2) Chase-acceleration, a catalytic cooperativity, requires at least two intact catalytic sites . 3) Single-site catalysis can be mediated by a single intact catalytic site alone . Rescrambling of subunits between complexes could occur when the complex was aged under certain conditions, and this might be one of the reasons for previous contradictory results (Miwa, K., Ohtsubo, M., Denda, K., Hisabori, T., Date, T., and Yoshida, M.(1989) J . Biochem . (Tokyo) 106, 730-734). Arch Microbiol, 1996 Jul 24, 166(1), 64 - 7 Culturability and survival of an extreme thermophile isolated from deep-sea hydrothermal vents Gonz&aacute;lez JM, Kato C, Horikoshi K. The culturability of a strictly anaerobic, extremely thermophilic archaeon, Thermococcus peptonophilus (optimal growth temperature: 85&deg; C), was studied during survival stages at various temperatures (98, 85, 70, and 4&deg; C) . Total cell number (determined by DAPI staining), active cells (rhodamine-stained cells), and culturable cells (using most-probable-number) were counted over time . The number of culturable cells decreased under each condition tested . The total number of cells significantly decreased only at temperatures close to the maximum for growth (98&deg; C); at this temperature, the cells spontaneously lysed . Our results suggested that survival at 4&deg; C in oxygenated waters might be a mechanism for the dispersion of extreme thermophiles in the ocean . In addition, we proved the existence of T . peptonophilus cells in several physiological states: culturable cells, active non-culturable cells, inactive non-culturable cells, and dead cells . Cell death was caused by cellular lysis. J Biol Chem, 1996 Jul 19, 271(29), 17343 - 8 A novel factor required for the assembly of the DnaK and DnaJ chaperones of Thermus thermophilus; Motohashi K et al.; We previously reported the isolation of T.DnaK.DnaJ chaperone complex from Thermus thermophilus . Here, we show that a novel factor is necessary for the assembly of T.DnaK and T.DnaJ into the complex . A dnaK gene cluster of T . thermophilus contained five genes, dnaK-grpE-dnaJ-orf4-clpB . Interestingly, T.DnaJ lacks the whole "cysteine-rich region" that has been postulated to be necessary to bind unfolded proteins . The orf4 gene encodes a novel 78-amino acid protein . Curiously, T.DnaK and T.DnaJ expressed in Escherichia coli did not form the complex . Careful reexamination of the T.DnaK.DnaJ complex revealed the presence of a small protein in the complex, which turned out to be a product of orf4 . As expected, expression of three genes, dnaK-dnaJ-orf4, resulted in production of a T.DnaK.DnaJ complex in E . coli that was indistinguishable from the authentic complex in its ability to interact with nucleotide and denatured protein . The product of orf4 was also required for in vitro reconstitution of the complex and named T.DafA (T.DnaK.DnaJ assembly factor A) . The complex comprises three copies each of T.DnaK, T.DnaJ, and T.DafA . Even though a definite homolog of T.DafA has not been found in the data base, this finding raises a possibility that interaction between DnaK and DnaJ chaperones in other organisms is also mediated by a small protein yet unnoticed. FEMS Microbiol Lett, 1996 Jul 15, 141(1), 37 - 43 Sequencing, cloning and expression of a beta-1,4-mannanase gene, manA, from the extremely thermophilic anaerobic bacterium, Caldicellulosiruptor Rt8B.4; Gibbs MD et al.; A gene encoding a beta-mannanase (manA) has been cloned from an obligately anaerobic extreme thermophile, Caldicellulosiruptor strain Rt8B.4, which is most closely related to Caldicellulosiruptor saccharolyticus (formerly Caldocellum saccharolyticum) . The gene codes for a multidomain enzyme with a C-terminal beta-mannanase domain which was amplified by the polymerase chain reaction and cloned into a temperature-inducible expression vector in Escherichia coli . Sequence comparisons have shown that the Man domain of Rt8B.4 ManA is related to a thermophilic Dictyoglomus mannanase and a mesophilic mannanase from a Bacillus species . It appears to be unrelated to the beta-mannanase domain of C . saccharolyticus, implying acquisition of the genes from unrelated sources by the two bacteria. Eur J Biochem, 1996 Jul 15, 239(2), 501 - 8 Asparaginyl-tRNA synthetase from Thermus thermophilus HB8 . Sequence of the gene and crystallization of the enzyme expressed in Escherichia coli; Seignovert L et al.; The gene for the asparaginyl-tRNA synthetase, a class IIb enzyme, from the extreme thermophile Thermus thermophilus HB8 has been cloned and sequenced . Sequence analysis revealed an open reading frame that codes for a protein of 438 amino acid residues (50875 Da) . Codon usage in the asparaginyl-tRNA synthetase gene (asnS) is similar to the characteristic usage in the genes for proteins from bacteria of the genus Thermus, and the G+C content in the third position of the codons is as high as 94% . The amino acid sequence of asparaginyl-tRNA synthetase from T . thermophilus shows high similarity with other bacterial asparaginyl-tRNA synthetase sequences (30-55% identity) . By expression of the T . thermophilus asnS gene in Escherichia coli, the thermostable enzyme was overproduced and purified to homogeneity by heat treatment and two chromatography steps . The protein obtained is remarkably thermostable and retains 50% of its initial tRNA aminoacylation activity after 1 h of incubation at 90 degrees C or 21 h at 85 degrees C . Crystals of the enzyme were obtained from polyethylene glycol 6000 solutions by vapour diffusion techniques . The crystals diffract X-rays beyond 2.8 A. Eur J Biochem, 1996 Jul 15, 239(2), 265 - 71 Limited proteolysis and amino acid replacements in the effector region of Thermus thermophilus elongation factor Tu; Zeidler W et al.; The effector region of the elongation factor Tu (EF-Tu) from Thermus thermophilus was modified by limited proteolysis or via site-directed mutagenesis . The biochemical properties of the obtained EF-Tu variants were investigated with respect to partial reactions of the functional cycle of EF-Tu . EF-Tu that was cleaved at the Arg59-Gly60 peptide bond {EF-Tu-(1-59)/EF-Tu-(60-405)} bound GDP, EF-Ts and aminoacyl-tRNA, had normal intrinsic GTPase activity and was active in poly(U)-dependent poly(Phe) synthesis . However, the GTPase activity of EF-Tu-(1-59)/EF-Tu-(60-405) was not stimulated by T . thermophilus 70S ribosomes, and its GTP-dissociation rate was increased compared with that of intact EF-Tu . EF-Tu cleaved at the Lys52-Ala53 peptide bond has properties similar to EF-Tu-(1-59)/EF-Tu-(60-405) . By means of site-directed mutagenesis, Glu55 was replaced by Leu, Glu56 by Ala and Arg59 by Thr in T . thermophilus EF-Tu . These amino acid substitutions did not substantially affect either the affinity of EF-Tu . GTP for aminoacyl-tRNA or the interactions with GDP, GTP or EF-Ts . Similarly the intrinsic GTPase activity is not influenced . Replacement of Glu56 by Ala led to strong reduction in the ribosome-induced GTPase activity . This effect is specific since replacement of the neighbouring Glu55 by Leu did not affect the ribosome-induced GTPase activity . The results demonstrate that the structure of the effector region of EF-Tu in the vicinity of Arg59 is important for the control of the GTPase activity by ribosomes. J Biol Chem, 1996 Jul 12, 271(28), 16559 - 66 Ribonuclease P of Tetrahymena thermophila; True HL et al.; Ribonuclease P (RNase P) is responsible for the generation of mature 5' termini of tRNA . The RNA component of this complex encodes the enzymatic activity in bacteria and is itself catalytically active under appropriate conditions in vitro . The role of the subunits in eucaryotes has not yet been established . We have partially purified RNase P activity from the ciliate protozoan Tetrahymena thermophila to learn more about the biochemical characteristics of RNase P from a lower eucaryote . The Tetrahymena RNase P displays a pH optimum and temperature optimum characteristic of RNase P enzymes isolated from other organisms . The Km of the T . thermophila enzyme for pre-tRNAGln is 1.6 x 10(-7)M, which is comparable to the values reported for other examples of RNase P . The Tetrahymena RNase P is a ribonucleoprotein complex, as supported by its sensitivity to micrococcal nuclease and proteinase K . The buoyant density of the enzyme in Cs2SO4 is 1.42 g/ml, which suggests that the RNA component of the Tetrahymena enzyme comprises a significantly greater percentage of the holoenzyme than that determined for RNase P of other Eucarya or Archaea . The holoenzyme has a requirement for divalent cations displaying characteristics that are unique for RNase P but closely resemble preferences reported for the Tetrahymena group I intron RNA . Puromycin inhibits pre-tRNA processing by the Tetrahymena complex, and implications of the similarities between recognition of tRNA by ribosomal components and RNase P are discussed. Biochem Biophys Res Commun, 1996 Jul 5, 224(1), 224 - 8 The effect of cysteine-43 mutation on thermostability and kinetic properties of citrate synthase from Thermoplasma acidophilum; Kocabiyik S et al.; In this study, we have substituted serine-43 by cysteine in the recombinant citrate synthase from a moderately thermophilic Archaeon Thermoplasma acidophilum, for site-specific attachment of labels and have investigated the effects of this mutation on the biochemical properties and thermal stability of the enzyme . Both wild-type and the mutant enzymes were purified to homogenity using affinity chromatography on Matrex Gel Red A . The mutant Thermoplasma citrate synthase is very similar to wild-type citrate synthase in its substrate and co-factor specificities, pH profile and thermal stability . The mutation, however, has decreased the enzyme activity . The newly introduced reactive sulphydryl group could be easily modified by DTNB and labelled with 4-chloro-7-sulphobenzofuran, without loss of any activity. Dtsch Tierarztl Wochenschr, 1996 Jul, 103(7), 264 - 8 {Effects of residues of anti-infective agents in animal excretions on slurry treatment and the soil}; Bohm R; Application of antibiotics and feed additives in animal husbandry generates populations of resistant bacteria in the gut which are introduced via slurry into the ecosystem soil . Nevertheless the influence to the ecosystem soil seems to be low, due to inactivation and dilution in slurry and soil . The probability that this pathway contributes to the problems of antibiotic resistance in human medicine is low . Aerobic and anaerobic treatment of slurry is more or less influenced by residuals of some feed additives and antibiotics . The aerobic-thermophilic treatment seems generally to be more sensitive than the anaerobic biogas production, except in some special cases . No evidence could be found that residuals of antibiotics in slurry have a negative influence on the ecosystem soil, due to preliminary results obtained by similar field trials with disinfectants . The application of antibiotics in aquaculture must be regarded more critically. Eur J Cell Biol, 1996 Jul, 70(3), 243 - 9 A giant protein associated with the anterior pole of a trypanosomatid cell body skeleton; Baqui MM et al.; A megadalton protein was found to be a cytoskeleton component of the promastigote forms of the flagellate Phytomonas serpens . This protein migrated on sodium dodecyl sulfate polyacrylamide gel electrophoresis as a doublet of polypeptides with a molecular mass similar to muscle beta-connectin (titin) 2500-3000 kDa . A polyclonal antibody raised against this protein reacts, by immunoblot analysis, with Phytomonas serpens and two others Phytomonas species . In addition, the Phytomonas serpens protein was immunoprecipitated after being metabolically labeled with {35S}methionine . This antibody did not cross-react with the cytoskeletal proteins of Trypanosoma cruzi, Crithidia luciliae thermophila, Crithidia fasciculata and Leptomonas samueli or with beta-connectin (titin) . Indirect immunofluorescence microscopy analysis revealed a punctate fluorescence staining at the anterior region of the parasite's body skeleton . Moreover, immunogold electron microscopy of cytoskeletal preparations and of thin sections of whole cells indicates that the giant protein appears to cap the anterior end of the cell body microtubules at the level of the junctional complex . We suggest that this giant protein may serve as a linker between the cell body skeleton and the flagellum membrane. Vet Microbiol, 1996 Jul, 51(1-2), 77 - 84 Rapid and specific detection of Mycoplasma agalactiae by polymerase chain reaction; Tola S et al.; A polymerase chain reaction (PCR)-based test was developed for the detection of Mycoplasma agalactiae in sheep milk samples . Two oligonucleotide primers were designed to amplify a 375 bp fragment of M . agalactiae chromosomal DNA . Amplified products were analyzed by agarose gel electrophoresis and Southern blot hybridization using a fluorescein labeled 528 bp probe . The primers allowed the amplification of fragment of M . agalactiae DNA and did not amplify any specific fragment of other mycoplasmal DNAs (M . capricolum, M . mycoides subsp . mycoides, M . mycoides subsp . capri, M . putrefaciens, M . arginini and M . bovis) or other bacterial DNAs (S . aureus, S . epidermidis, P . haemalytica, E . coli, S . agalactiae, S . dysgalactiae, S . uberis, B . cereus, P . aeruginosa, S . durans, L . lactis, L . lactis var . diacetilactis, L . mesenteroides, S . thermophilus, L . bulgaricus and L . casei) . The limit of detection of PCR assay was between 2.5 and 25 fg of purified DNA and 10(2) CCU ml-1 on mycoplasma cultures . These results indicate that the PCR technique can be used as a rapid and specific diagnostic method for detection of M . agalactiae. Int J Syst Bacteriol, 1996 Jul, 46(3), 727 - 35 Phylogeny and taxonomy of mesophilic Methanococcus spp . and comparison of rRNA, DNA hybridization, and phenotypic methods; Keswani J et al.; The phylogeny and taxonomy of the mesophilic methane-producing archaea of the order Methanococcales were examined by DNA relatedness, 16S rRNA sequence analysis, cellular protein patterns, and phenotypic methods . The mesophilic species Methanococcus maripaludis, Methanococcus vannielii, Methanococcus voltaei, and "Methanococcus aeolicus" formed a deep group with 5 to 30% DNA relatedness and 92 to 96% 16S rRNA sequence similarity . Twenty-two additional isolates and Methanococcus deltae were similar to the type strain of either M . voltaei or M . maripaludis . Two isolates, strains A2 and A3, exhibited 37% DNA relatedness and 99.2% 16S rRNA sequence similarity to M . voltaei PS(T) (T = type strain) . In the absence of phenotypic differences, these organisms were assigned to M . voltaei . Similarly, four autotrophic isolates, strains C5, C6, C7, and C8, exhibited 54 to 69% DNA relatedness and 99.2% 16S rRNA sequence similarity to M . maripaludis JJT and were assigned to M . Maripaludis . While these isolates were sufficiently genetically diverse to justify classification in novel species, few differences were apparent in the phenotypic properties available for measurement . Thus, the phenotypic properties of these lithotrophic archaea were highly conserved and poor indicators of genetic diversity . Partial sequencing of about 200 bases of both the 16S and 23S rRNAs of the isolates demonstrated allelic diversity within methanococcal species . This allelic diversity did not correlate with diversity measured by DNA relatedness, cellular protein pattern, and other methods . Similarly, antisera to whole cells of the type strains did not cross-react strongly to whole cells of strains that were genetically similar, and serological cross-reactivity was not a useful taxonomic method for methanococci . Lastly, on the basis of the results of 16S rRNA sequence analyses and biochemical data, the ancestor of the mesophilic methanococci may have been an autotrophic thermophile. Appl Environ Microbiol, 1996 Jul, 62(7), 2657 - 9 L-alanine production from glucose fermentation by hyperthermophilic members of the domains bacteria and Archaea: a remnant of an ancestral metabolism? Ravot G, Ollivier B, Fardeau ML, Patel BK, Andrews KT, Magot M, Garcia JL. New members of the order Thermotogales were isolated from nonvolcanically heated geothermal environments, including oil fields and waters of the Great Artesian Basin of Australia, thereby extending their known habitats, previously recognized primarily as volcanic . The hyperthermophilic and thermophilic members of Thermotogales of volcanic origin, together with the recently described nonvolcanic species of this order and three new isolates described in this paper, were all found to produce L-alanine from glucose fermentation, in addition to acetate, lactate, CO2 and H2 . L-alanine production from glucose is a trait in common with Pyrococcus furiosus and Thermococcus profundus . We propose that L-alanine production from sugar fermentation be regarded as an ancestral metabolic characteristic. Appl Environ Microbiol, 1996 Jul, 62(7), 2482 - 8 Purification and characterization of a fibrinolytic enzyme produced from Bacillus sp . strain CK 11-4 screened from Chungkook-Jang; Kim W et al.; Bacillus sp . strain CK 11-4, which produces a strongly fibrinolytic enzyme, was screened from Chungkook-Jang, a traditional Korean fermented-soybean sauce . The fibrinolytic enzyme (CK) was purified from supernatant of Bacillus sp . strain CK 11-4 culture broth and showed thermophilic, hydrophilic, and strong fibrinolytic activity . The optimum temperature and pH were 70 degrees C and 10.5, respectively, and the molecular weight was 28,200 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis . The first 14 amino acids of the N-terminal sequence of CK are Ala-Gin-Thr-Val-Pro-Tyr-Gly-Ile-Pro-Leu-Ile-Lys-Ala-Asp . This sequence is identical to that of subtilisin Carlsberg and different from that of nattokinase, but CK showed a level of fibrinolytic activity that was about eight times higher than that of subtilisin Carlsberg . The amidolytic activity of CK increased about twofold at the initial state of the reaction when CK enzyme was added to a mixture of plasminogen and substrate (H-D-Val-Leu-Lys-pNA) . A similar result was also obtained from fibrin plate analysis. FEMS Microbiol Lett, 1996 Jul 1, 140(2-3), 271 - 6 A novel thermostable dextranase from a Thermoanaerobacter species cultured from the geothermal waters of the Great Artesian Basin of Australia; Wynter C et al.; A Gram-negative sporulating thermophilic anaerobe, designated AB11Ad, was isolated from the heated waters of the Great Artesian Basin of Australia . It grew on a variety of carbohydrates including glucose, starch, and dextran and produced a thermostable and thermoactive extracellular endo-dextranase . The enzyme was produced more actively under pH controlled continuous culture conditions than under batch conditions . Ammonium sulfate precipitated crude dextranase exhibited a temperature optimum of 70 degrees C and a pH optimum between 5 and 6 . The half life was approximately 6.5 h at 75 degrees C and 2 h at 80 degrees C at pH 5.0 and in the absence of added dextran . 16S rRNA sequence analysis indicated that isolate AB11Ad was a member of the genus Thermoanaerobacter. Microbiology, 1996 Jul, 142 ( Pt 7), 1651 - 7 The Bacillus stearothermophilus NUB36 surA gene encodes a thermophilic sucrase related to Bacillus subtilis SacA; Li Y et al.; The complete nucleotide sequence of the surA gene, encoding a sucrase from Bacillus stearothermophilus NUB36, was determined . surA was composed of 1338 bp and encoded 445 amino acid residues . The deduced polypeptide of M(r) 51519 showed strong sequence similarity to sucrose and sucrose phosphate hydrolases from Bacillus subtilis, Klebsiella pneumoniae and Vibrio alginolyticus, and contained the 'sucrose box' residues thought to be important for catalysis of the transfer of fructose from sucrose . The enzyme was partially purified using affinity chromotography from extracts of Escherichia coli containing the cloned surA . SurA displayed an optimum temperature for sucrose hydrolysis of 55 degrees C and high stability . The M(r) of SurA determined by gel filtration was 105,000, which suggested that the active form of the enzyme is a dimer . SurA exhibited an apparent Km of 40 mM for sucrose but, unlike the homologous B . subtilis enzyme, had no detectable sucrose phosphate hydrolase activity. DNA Cell Biol, 1996 Jul, 15(7), 589 - 94 Fidelity and predominant mutations produced by deep vent wild-type and exonuclease-deficient DNA polymerases during in vitro DNA amplification; Huang H et al.; Denaturing gradient gel electrophoresis (DGGE) was used to examine error rates and mutations induced by native (wt) and exonuclease-deficient (exo-) Deep Vent DNA polymerases during DNA amplification by polymerase chain reaction (PCR), in the presence or absence of the T4 bacteriophage gene 32 protein (gp32).gp32 was found to decrease the error rate of the wt, but not that of the exo-, Deep Vent . The average errors per base duplication for the native form were 8.0 x 10(-5) and 6.0 x 10(-5) in the absence and presence of gp32, respectively . For the exo- form, the error rates were 2.0 x 10(-4) and 2.2 x 10(-4) errors per base duplication in the absence and presence of gp32, respectively . Examination of mutations produced by native Deep Vent showed that A/T to G/C transition predominated, consistent with the results of our earlier studies with DNA polymerases derived from other thermophilic bacteria . These results indicate that PCR with high fidelity can be achieved by using wt Deep Vent in combination with gp32. Eur J Biochem, 1996 Jul 1, 239(1), 93 - 7 Si-face stereospecificity at C5 of coenzyme F420 for F420-dependent glucose-6-phosphate dehydrogenase from Mycobacterium smegmatis and F420-dependent alcohol dehydrogenase from Methanoculleus thermophilicus; Klein AR et al.; Coenzyme F420 is a 5-deazaflavin . Upon reduction, 1,5-dihydro-coenzyme F420 is formed with a prochiral center at C5 . In this study we report that the F420-dependent glucose-6-phosphate dehydrogenase from Mycobacterium smegmatis and the F420-dependent alcohol dehydrogenase from Methanoculleus thermophilicus are Si-face stereospecific with respect to C5 of the 5-deazaflavin . These results were obtained by following the stereochemical course of the reversible incorporation of 3H into F420 from tritium-labeled substrates . Our findings bring to eight the number of coenzyme-F420-dependent enzymes shown to be Si-face stereospecific . No F420-dependent enzyme with Re-face stereospecificity is known . This is noteworthy since coenzyme F420 is functionally similar to pyridine nucleotides for which both Si-face and Re-face specific enzymes have been found. Eur J Biochem, 1996 Jul 1, 239(1), 150 - 5 Purification and characterisation of an alpha-glucan phosphorylase from the thermophilic bacterium Thermus thermophilus; Boeck B et al.; An alpha-glucan phosphorylase has been purified 4500-fold from the thermophilic bacteria Thermus thermophilus . In contrast to other bacterial phosphorylases the thermophilic enzyme seems neither to be inducible by maltose nor repressed by glucose . T . thermophilus phosphorylase shares major properties with known mesophilic phosphorylases such as pyridoxal 5'-phosphate content (1 M pyridoxal-P/M subunit), subunit molecular mass (about 90 kDa) and inhibitor constants . The optimum temperature of T . thermophilus phosphorylase was observed at 70 degrees C in the pH range 5.5-6.5 . While at 25 degrees C the subunit composition of the thermophilic enzyme is an octameric form, the preferential form at the optimum temperature of 70 degrees C seems to be a dimer . Most remarkably, in the direction of synthesis and degradation the limiting size of the oligosaccharide substrate is shorter by one glucose residue than the minimum size of substrate degraded by other alpha-glucan phosphorylases . Maltotetraose and glycogen are degraded with rates similar to that observed with maltoheptaose (Vmax = 18 U/mg) . Correspondingly, maltotriose functions as primer in the synthesis direction . Differences in fluorescence and absorption spectra of the cofactor and the failure of arsenate acting as a substrate indicate that the active site structure of T . thermophilus phosphorylase differs from that of known alpha-glucan phosphorylases. Biochem J, 1996 Jul 1, 317 ( Pt 1), 29 - 34 Molecular cloning of delta 9 fatty acid desaturase from the protozoan Tetrahymena thermophila and its mRNA expression during thermal membrane adaptation; Nakashima S et al.; In response to a decrease in its growth temperature, the protozoan Tetrahymena is known to increase the level of unsaturated fatty acids in its membrane phospholipids so as to maintain the correct physical state (fluidity) of the membranes . In this organism, synthesis of unsaturated fatty acids is initiated by delta 9 acyl-CoA desaturase . Our previous studies have shown that, during cold adaptation, the activity of microsomal palmitoyl- and stearoyl-CoA desaturase increases, reaching a maximal level at 2 h after a temperature down-shift to 15 degrees C . Two hypotheses have been proposed to explain this increase in desaturase activity: (1) self-regulation via a direct effect of reduced membrane fluidity, and (2) induction of desaturase mRNA . However, the precise mechanism is not clearly understood . In order to obtain further insight into the mechanism of regulation of the desaturase, we have isolated a gene that encodes delta 9 fatty acid desaturase from T . thermophila and examined its expression during cold adaptation . The nucleotide sequence indicates that the 1.4 kbp gene encodes a polypeptide of 292 amino acid residues which shows marked sequence similarity to delta 9 acyl-CoA desaturases from other sources, e.g . rat, mouse, Amblyomma americanum and Saccharomyces cerevisiae . This protein has three histidine-cluster motifs (one HXXXXH and two HXXHH), and two hydrophobic regions which are conserved among delta 9 acyl-CoA desaturases . The level of desaturase mRNA was sensitive to decreasing the temperature of the culture media, and was close to maximal immediately after the temperature was shifted down from 35 degrees C to 15 degrees C (0.8 degrees C/min) . Thereafter, the amount of mRNA gradually decreased with time, but remained above the control level for at least 5 h . Furthermore, during the course of the cooling process to 15 degrees C, the increased expression of desaturase mRNA became evident at 27 degrees C . Nuclear run-on analysis and actinomycin D chase experiments revealed that the elevation of the mRNA level was due to increases in both transcription and mRNA stability . These results suggest that the enhanced desaturase activity is controlled, at least in part, at the transcriptional level. Biochem J, 1996 Jul 1, 317 ( Pt 1), 235 - 45 Tetrameric malate dehydrogenase from a thermophilic Bacillus: cloning, sequence and overexpression of the gene encoding the enzyme and isolation and characterization of the recombinant enzyme; Wynne SA et al.; The gene encoding the tetrameric malate dehydrogenase (MDH) in a thermophilic Bacillus species (BI) has been cloned in an Escherichia coli plasmid . The nucleotide sequence of the gene, the first to be elucidated for a tetrameric MDH, shows the MDH subunit to contain 312 amino acids and have a molecular mass of 33648 Da, which confirms the experimentally determined value of about 35 kDa . Like the genomic DNA of BI, the MDH gene is relatively AT-rich; this contrasts with the generally GC-rich nature of the DNA of thermophilic Bacillus species . Comparison of amino acid sequences reveals that BI MDH bears greater structural similarity to lactate dehydrogenases (LDHs) than to other (dimeric) MDHs . MDHs and LDHs resemble each other in catalytic mechanism and several other respects . However, whereas MDHs in the majority of organisms are dimers, the tetrameric structure is favoured among LDHs . The stronger structural resemblance that BI MDH has to LDHs than to the dimeric MDHs provides some explanation as to why Bacillus MDH, unlike most other MDHs, is tetrameric . A 1 kb fragment containing the BI MDH gene, produced in a PCR, has been cloned into a high-expression E . coli plasmid vector . BI MDH synthesized from this clone constitutes about 47% of the total protein in cell extracts of the E . coli strain carrying the clone . MDH purified from BI and that purified from the E . coli strain carrying the MDH gene clone appear to be identical proteins by several criteria . A number of characteristics of the MDH have been elucidated, including the molecular masses of the native enzyme and the subunit, N-terminal amino acid sequence, isoelectric point, pH optimum for activity, thermostability, stability to pH, urea and guanidinium chloride and several kinetic parameters . Whereas the MDH is a stable tetramer in the pH range 5-7, it appears to be converted into a stable dimer at pH 3.5 . This suggests that the dimer is a stable intermediate in the dissociation of the tetramer to monomers at low pH. EMBO J, 1996 Jul 1, 15(13), 3286 - 95 Mitochondrial import of only one of three nuclear-encoded glutamine tRNAs in Tetrahymena thermophila; Rusconi CP et al.; The mitochondrial genome of Tetrahymena does not appear to encode enough tRNAs to perform mitochondrial protein synthesis . It has therefore been proposed that nuclear-encoded tRNAs are imported into the mitochondria . T.thermophila has three major glutamine tRNAs: tRNA(Gln)(UUG), tRNA(Gln)(UUA) and tRNA(Gln)(CUA) . Each of these tRNAs functions in cytosolic translation . However, due to differences between the Tetrahymena nuclear and mitochondrial genetic codes, only tRNA(Gln)(UUG) has the capacity to function in mitochondrial translation as well . Here we show that approximately 10-20% of the cellular complement of tRNA(Gln)(UUG) is present in mitochondrial RNA fractions, compared with 1% or less for the other two glutamine tRNAs . Furthermore, this glutamine tRNA is encoded only by a family of nuclear genes, the sequences of several of which are presented . Finally, when marked versions of tRNA(Gln)(UUG) and tRNA(Gln)(UUA) flanked by identical sequences are expressed in the macronucleus, only the former undergoes mitochondrial import; thus sequences within tRNA(Gln)(UUG) direct import . Because tRNA(Gln)(UUG) is a constituent of mitochondrial RNA fractions and is encoded only by nuclear genes, and because ectopically expressed tRNA(Gln)(UUG) fractionates with mitochondria like its endogenous counterpart, we conclude that it is an imported tRNA in T.thermophila. Mol Cell Biol, 1996 Jul, 16(7), 3720 - 9 The testis-specific high-mobility-group protein, a phosphorylation-dependent DNA-packaging factor of elongating and condensing spermatids; Alami-Ouahabi N et al.; Mammalian spermiogenesis is characterized by a striking restructuring of the spermatid chromatin caused by the replacement of nucleohistones with transition proteins and their subsequent replacement with nucleoprotamines . The onset of nuclear elongation and chromatin condensation in spermatids is accompanied by a general decrease in the transcriptional activity of the DNA . A recently identified testis-specific high-mobility-group (tsHMG) protein, similar to the human mitochondrial transcription factor I and to the linker-associated protein delta of Tetrahymena thermophila micronuclei, is thought to play a structural role in this process . We confirm by immunoblot analysis of fractionated germ cells that the presence of tsHMG is restricted to transcriptionally quiescent elongating and condensing spermatids . Purified recombinant tsHMG protein displays preferential binding to supercoiled plasmid DNA, which reversibly protects the DNA against the DNA-relaxing activity of eukaryotic topoisomerase I and also impairs the transcriptional activity of this template when assayed in vitro . The tsHMG protein can also introduce negative supercoils into a relaxed plasmid substrate in a topoisomerase I-dependent manner . We also show that the tsHMG protein is the substrate of a Ca2+-phospholipid-dependent protein kinase (protein kinase C) present in testis extracts of adult mice and demonstrate that phosphorylation by protein kinase C is required for both the DNA-binding and the topoisomerase I-dependent supercoiling activities of tsHMG . Our results support the hypothesis that the spermatid tsHMG protein is a topological factor (transition protein) that can modulate the activity of topoisomerase I . This activity could contribute to the important transition in chromatin structure which leads to the decrease in DNA metabolism observed at the early stages of spermatid elongation. Mol Cell Biol, 1996 Jul, 16(7), 3658 - 67 Non-Mendelian, heritable blocks to DNA rearrangement are induced by loading the somatic nucleus of Tetrahymena thermophila with germ line-limited DNA; Chalker DL et al.; Site-specific DNA deletion occurs at thousands of sites within the genome during macronuclear development of Tetrahymena thermophila . These deletion elements are usually not detected in macronuclear chromosomes . We have interfered with the normal deletion of two of these elements, the adjacent M and R elements, by loading vegetative macronuclei with these elements prior to sexual conjugation . Transformed cell lines containing the exogenous M or R element, carried on high-copy-number vectors containing genes encoding rRNA within parental (old) macronuclei, consistently failed to excise chromosomal copies of the M or R element during formation of new macronuclei . Little or no interference with the deletions of adjacent elements or of unlinked elements was observed . The micronucleus (germ line)-limited region of each element was sufficient to inhibit specific DNA deletion . This interference with DNA deletion usually is manifested as a cytoplasmic dominant trait: deletion elements present in the old macronucleus of one partner of a mating pair were sufficient to inhibit deletion occurring in the other partner . Remarkably, the failure to excise these elements became a non-Mendelian, inheritable trait in the next generation and did not require the high copy number of exogenously introduced elements . The introduction of exogenous deletion elements into parental macronuclei provides us with an epigenetic means to establish a heritable pattern of DNA rearrangement. J Biol Chem, 1996 Jun 28, 271(26), 15358 - 66 Unidirectional reconstitution into detergent-destabilized liposomes of the purified lactose transport system of Streptococcus thermophilus; Knol J et al.; The lactose transport protein (LacS) of Streptococcus thermophilus was amplified to levels as high as 8 and 30% of total membrane protein in Escherichia coli and S . thermophilus, respectively . In both organisms the protein was functional and the expression levels were highest with the streptococcal lacS promoter . Also a LacS deletion mutant, lacking the carboxyl-terminal regulatory domain, could be amplified to levels >20% of membrane protein . Membranes from S . thermophilus proved to be superior in terms of efficient solubilization and ease and extent of purification of LacS; >95% of LacS was solubilized with relatively low concentrations of Triton X-100, n-octyl-beta-D-glucoside, n-dodecyl-beta-D-maltoside, or C12E8 . The LacS protein carrying a poly-histidine tag was purified in large quantities (approximately 5 mg/liter of culture) and with a purity >98% in a two-step process involving nickel chelate affinity and anion exchange chromatography . The membrane reconstitution of LacS was studied systematically by stepwise solubilization of preformed liposomes, prepared from E . coli phospholipid and phosphatidylcholine, and protein incorporation at the different stages of liposome solubilization . The detergents were removed by adsorption onto polystyrene beads and H+-lactose symport and lactose counterflow were measured . Highest transport activities were obtained when Triton X-100 was used throughout the solubilization/purification procedure, whereas activity was lost irreversibly with n-octyl-beta-D-glucoside . For reconstitutions mediated by n-dodecyl-beta-D-maltoside, C12E8, and to a lesser extent Triton X-100, the highest transport activities were obtained when the liposomes were titrated with low amounts of detergent (onset of liposome solubilization) . Importantly, under these conditions proteoliposomes were obtained in which LacS was reconstituted in an inside-out orientation, as suggested by the outside labeling of a single cysteine mutant with a membrane impermeable biotin-maleimide . The results are consistent with a mechanism of reconstitution in which the hydrophilic regions of LacS prevent a random insertion of the protein into the membrane . Consistent with the in vivo lactose/galactose exchange catalyzed by the LacS protein, the maximal rate of lactose counterflow was almost 2 orders of magnitude higher than that of H+-lactose symport. Biochemistry, 1996 Jun 18, 35(24), 7812 - 8 Isolation and characterization of soluble electron transfer proteins from Chromatium purpuratum; Kerfeld CA et al.; Several soluble electron transfer proteins were isolated and characterized from the marine purple-sulfur bacterium Chromatium purpuratum . The C . purpuratum flavocytochrome c is similar in molecular mass (68 kDa) and isoelectric point (6.5) to flavocytochromes isolated from other phototrophs . Redox titrations of the flavocytochrome c hemes show two components with midpoint potential values of +15 and -120 mV, behavior similar to that observed with the flavocytochrome isolated from the thermophilic Chromatium tepidum . Moreover, N-terminal amino acid sequence analysis of both the flavin and the cytochrome subunit indicates substantial homology to the primary structure of the flavocytochrome c of Chromatium vinosum . In contrast, the C . purpuratum high-potential iron-sulfur protein (HiPIP) differs from those isolated from other photosynthetic bacteria in its relatively high midpoint potential (+390 mV) and the possibility that it exists as a dimer in solution . Two low molecular mass c-type cytochromes were also characterized . One appears to be a high-potential (+310 mV) c8-type cytochrome . Amino acid sequencing suggests that the second cytochrome may be a homologue of the low-potential cytochrome c-551, previously described in two species of Ectothiorhodospirillaceae. Nucleic Acids Res, 1996 Jun 15, 24(12), 2429 - 34 Factors affecting fidelity of DNA synthesis during PCR amplification of d(C-A)n.d(G-T)n microsatellite repeats; Hite JM et al.; The susceptibility of microsatellite DNA sequences to insertions and deletions in vivo makes them useful for genetic mapping and for detecting genomic instability in tumors . An in vitro manifestation of this instability is the production of undesirable frameshift products during amplification of (dC-dA)n x (dG-dT)n microsatellites in the polymerase chain reaction (PCR) . These products differ from the primary product by multiples of 2 nucleotides . We have tested the hypothesis that factors known to affect the fidelity of DNA synthesis may affect (dC-dA)n x (dG-dT)n frameshifting during the PCR . Neither modifications of pH, dNTP concentration, and Mg++ concentration using Amplitaq, nor the use of thermophilic DNA polymerases including UITma, Pfu, Vent and Deep Vent significantly decreased the production of frameshift products during amplification . However, 3'-->5' exonuclease activity in thermophilic DNA polymerases inhibited the accumulation of PCR products containing non-templated 3' terminal nucleotides . Most interestingly, extension temperatures of 37 degrees C during amplification using the thermolabile DNA polymerases Sequenase 1.0, Sequenase 2.0, and 3'-->5' exonuclease-deficient Klenow fragment greatly decreased the production of frameshift products . This method can improve the resolution of heterozygous or mutant (dC-dA)n x (dG-dT)n alleles differing in size by one or two repeat units. J Biol Chem, 1996 Jun 14, 271(24), 13987 - 92 The caa3 terminal oxidase of Bacillus stearothermophilus . Transient spectroscopy of electron transfer and ligand binding; Giuffre A et al.; The thermophilic bacterium Bacillus stearothermophilus possesses a caa3-type terminal oxidase, which was previously purified (De Vrij, W., Heyne, R . I . R., and Konings, W . N . (1989) Eur . J . Biochem . 178, 763-770) . We have carried out extensive kinetic experiments on the purified enzyme by stopped-flow time-resolved optical spectroscopy combined with singular value decomposition analysis . The results indicate a striking similarity of behavior between this enzyme and the electrostatic complex between mammalian cytochrome c and cytochrome c oxidase . CO binding to fully reduced caa3 occurs with a second order rate constant (k = 7.8 x 10(4)M-1 s-1) and an activation energy (E* = 6.1 kcal mol-1) similar to those reported for beef heart cytochrome c oxidase . Dithionite reduces cytochrome a with bimolecular kinetics, while cytochrome a3 (and CuB) is reduced via intramolecular electron transfer . When the fully reduced enzyme is mixed with O2, cytochrome a3, and cytochrome c are rapidly oxidized, whereas cytochrome a remains largely reduced in the first few milliseconds . When cyanide-bound caa3 is mixed with ascorbate plus TMPD, cytochrome c and cytochrome a are synchronously reduced; the value of the second order rate constant (k = 3 x 10(5) M-1 s-1 at 30 degrees C) suggests that cytochrome c is the electron entry site . Steady-state experiments indicate that cytochrome a has a redox potential higher than cytochrome c . The data from the reaction with O2 reveal a remarkable similarity in the kinetic, equilibrium, and optical properties of caa3 and the electrostatic complex cytochrome c/cytochrome c oxidase. Gene, 1996 Jun 12, 172(1), 49 - 51 BstF5I, an unusual isoschizomer of FokI; Abdurashitov MA et al.; BstF5I, a new restriction endonuclease (ENase) from Bacillus stearothermophilus F5, has been discovered . This enzyme recognizes 5'-GGATG-3' and cleaves DNA, generating a 2-base 3'extension: 5'-GGATG NN{symbol: see text}-3' 3'-CCTAC{symbol: see text}NN-5' BstF5I is an isoschizomer of FokI and seems to be evolutionarily close to other nonpalindromic-recognizing ENases from thermophilic bacilli. Biochemistry, 1996 Jun 11, 35(23), 7447 - 58 Identity of prokaryotic and eukaryotic tRNA(Asp) for aminoacylation by aspartyl-tRNA synthetase from Thermus thermophilus; Becker HD et al.; The aspartate identity of tRNA for AspRS from Thermus thermophilus has been investigated by kinetic analysis of the aspartylation reaction of different tRNA molecules and their variants as well as of tRNAPhe variants with transplanted aspartate identity elements . It is shown that G10, G34, U35, C36, C38, and G73 determine recognition and aspartylation of yeast and T.thermophilus tRNA(Asp) by the thermophilic AspRS . This set of nucleotides specifies also tRNA aspartylation in the homologous yeast and Escherichia coli systems . Structural considerations indicate that the major aspartate identity elements interact with amino acids conserved in all AspRSs . It follows that the structural features of tRNA and synthetase specifying aspartylation are mainly conserved in various structural contexts and in organisms adapted to different life conditions . Mutations of tRNA identity elements provoke drastic losses of charging in the heterologous system involving yeast tRNA(Asp) and T . thermophilus AspRS . In the homologous systems, the mutational effects are less pronounced . However, effects in E . coli and T . thermophilus exceed those in yeast which are particularly moderate, indicating variations in the individual contributions of identity elements for aspartylation in prokaryotes and eukaryotes . Analysis of multiple tRNA mutants reveals cooperativity between the cluster of determinants of the anticodon loop and the additional determinants G10 and G73 for efficient aspartylation in the thermophilic system, suggesting that conformational changes trigger formation of the functional tRNA/synthetase complex. EMBO J, 1996 Jun 3, 15(11), 2858 - 69 Developmentally programmed DNA deletion in Tetrahymena thermophila by a transposition-like reaction pathway; Saveliev SV et al.; We provide a molecular description of key intermediates in the deletion of two internal eliminated sequences (IES elements), the M and R regions, during macronuclear development in Tetrahymena thermophila . Using a variety of PCR-based methods in vivo, double-strand breaks are detected that are generated by hydrolytic cleavage and correspond closely to the observed chromosomal junctions left behind in the macronuclei . The breaks exhibit a temporal and structural relationship to the deletion reaction that provides strong evidence that they are intermediates in the deletion pathway . Breaks in the individual strands are staggered by 4 bp, producing a four nucleotide 5' extension . Evidence is presented that breaks do not occur simultaneously at both ends . The results are most consistent with a deletion mechanism featuring initiation by double-strand cleavage at one end of the deleted element, followed by transesterification to generate the macronuclear junction on one DNA strand . An adenosine residue is found at all the nucleophilic 3' ends used in the postulated transesterification step . Evidence for the transesterification step is provided by detection of a 3' hydroxyl that would be liberated by such a step at a deletion boundary where no other DNA strand ends are detected. EMBO J, 1996 Jun 3, 15(11), 2843 - 9 Variant minihelix RNAs reveal sequence-specific recognition of the helical tRNA(Ser) acceptor stem by E.coli seryl-tRNA synthetase; Saks ME et al.; Aminoacylation rate determinations for a series of variant RNA minihelix substrates revealed that Escherichia coli seryl-tRNA synthetase (SerRS) recognizes the 1--72 through 5--68 base pairs of the E.coli tRNA(Ser) acceptor stem with the major recognition elements clustered between positions 2--71 and 4--69 . The rank order of effects of canonical base pair substitutions at each position on kcat/Km was used to assess the involvement of major groove functional groups in recognition . Conclusions based on the biochemical data are largely consistent with the interactions revealed by the refined structure of the homologous Thermus thermophilus tRNA(Ser)-SerRS complex that Cusack and colleagues report in the accompanying paper . Disruption of an end-on hydrophobic interaction between the major groove C5(H) of pyrimidine 69 and an aromatic side chain of SerRS is shown to significantly decrease kcat/Km of a minihelix substrate . This type of interaction provides a means by which proteins can recognize the binary information of 'degenerate' sequences, such as the purine-pyrimidine base pairs of tRNA(Ser) . The 3--70 base pair is shown to contribute to recognition by SerRS even though it is not contacted specifically by the protein . The latter effect derives from the organization of the specific contacts that SerRS makes with the neighboring 2--71 and 4--69 acceptor stem base pairs. EMBO J, 1996 Jun 3, 15(11), 2834 - 42 The crystal structure of the ternary complex of T.thermophilus seryl-tRNA synthetase with tRNA(Ser) and a seryl-adenylate analogue reveals a conformational switch in the active site; Cusack S et al.; The low temperature crystal structure of the ternary complex of Thermus thermophilus seryl-tRNA synthetase with tRNA(Ser) (GGA) and a non-hydrolysable seryl-adenylate analogue has been refined at 2.7 angstrom resolution . The analogue is found in both active sites of the synthetase dimer but there is only one tRNA bound across the two subunits . The motif 2 loop of the active site into which the single tRNA enters interacts within the major groove of the acceptor stem . In particular, a novel ring-ring interaction between Phe262 on the extremity of this loop and the edges of bases U68 and C69 explains the conservation of pyrimidine bases at these positions in serine isoaccepting tRNAs . This active site takes on a significantly different ordered conformation from that observed in the other subunit, which lacks tRNA . Upon tRNA binding, a number of active site residues previously found interacting with the ATP or adenylate now switch to participate in tRNA recognition . These results shed further light on the structural dynamics of the overall aminoacylation reaction in class II synthetases by revealing a mechanism which may promote an ordered passage through the activation and transfer steps. Cell Biol Int, 1996 Jun, 20(6), 437 - 44 Insulin produces a biphasic response in Tetrahymena thermophila by stimulating cell survival and activating proliferation in two separate concentration intervals; Christensen ST et al.; Cells of Tetrahymena may produce autocrine signal molecules with effects on survival and proliferation . Here we have tested the effects of human recombinant and bovine insulin, and the B22-B30 fragment of bovine insulin over a wide range of concentrations (10(-5)-10(-18) M) on cell survival and proliferation in a synthetic nutrient medium . The cells were grown in conical flasks at low initial cell densities (40 and 400 cells/ml) . Insulin prevented rapid cell death and/or promoted cell proliferation over two separate concentration ranges: down to nanomolar levels and again in the low pico- and femtomolar range . At an initial population density of 400 cells/ml the cells multiplied at both concentration intervals . At 40 or fewer organisms/ml the cells multiplied in the high concentration interval, whereas in the low interval they survived for about four times longer than those in the control cultures . B22-B30 added to cultures of 40 initial cells/ml produced a stimulation of cell survival in the low pico- and high femtomolar range . In the presence of hemin (50 nM) cells at 400 initial organisms/ml multiplied at insulin concentrations down to about 3 nM and again from 300 am to 10 pM . In some cases, hemin plus insulin activated cell proliferation between the two concentration intervals as well . At 40 cells/ml the cells not only survived but proliferated in the femtomolar range . Cells in cultures supplemented with both hemin and B22-B30 multiplied at the low concentration interval (from about 100 fM to 10 pM). Biol Chem Hoppe Seyler, 1996 Jun, 377(6), 343 - 56 Glycyl-tRNA synthetase; Freist W et al.; Glycyl-tRNA synthetase, a class II aminoacyl-tRNA synthetase, catalyzes the synthesis of glycyl-tRNA, which is required to insert glycine into proteins . In a side reaction the enzyme also synthesizes dinuceloside polyphosphates, which probably participate in regulation of cell functions . Glycine is the smallest amino acid occurring in natural proteins, probably established as a protein component very early in evolution . Besides the amino and the carboxyl groups there is no functional group in the molecule . Alanine, the amino acid which is structurally most similar to glycine, possesses an additional methyl group as 'side chain' . Glycyl-tRNA synthetase is one of the few synthetases which exhibit different oligomeric structures in different organisms (alpha 2 beta 2 and alpha 2) . The alpha 2 beta 2 enzymes exhibit similarities to PheRS (also an alpha 2 beta 2 enzyme) . The alpha 2 forms belong to the subclass IIa enzymes with regard to sequence homologies . In eukaryotes the polypeptide is weakly associated with multienzyme complexes consisting of aminoacyl-tRNA synthetases . In the aminoacylation reaction a 'half-of-the-sites' mechanism as found for GlyRS from Bombyx mori is probably used by all glycyl-tRNA synthetases under in vivo conditions . Essentially, tRNAGly is recognized by GlyRS through standard identity elements in the anticodon region and in the acceptor stem . The last three facts may indicate that GlyRS is an enzyme which still possesses properties of a primordial aminoacyl-tRNA synthetase . Nine genes of glycyl-tRNA synthetases from six organisms have been sequenced . They encode synthetase subunits of chain lengths ranging from 300-700 amino acids . One crystal structure, that of the alpha 2 enzyme from Thermus thermophilus, has also been determined . The two subunits each possess three domains: the active site resembling that of aspartyl and seryl enzymes, a C-terminal anticodon recognition domain, and one domain which almost certainly interacts with the acceptor stem of tRNAGly . Antibodies against glycyl-RNA synthetase occur in the sera of patients suffering from polymyositis and interstitial lung disease. J Dairy Sci, 1996 Jun, 79(6), 943 - 55 Performance of commercial cultures in fluid milk applications; Sanders ME et al.; Six Lactobacillus acidophilus, 5 Bifidobacterium, and 6 Streptococcus thermophilus strains were studied for characteristics that are important to activity and stability in unfermented fluid milk products . Speciation, strain relatedness, frozen concentrate stability, bile sensitivity, and lactase activity were evaluated . The microbiological stability of a culture-containing fluid milk product was also determined . Two of the bifidobacteria cultures contained > 1 strain . Some strains were shown to be closely related or identical by pulsed-field gel electrophoresis of fragmented chromosomal DNA . Selective media that distinguished among all 3 added genera were identified . All lactobacilli and most of the bifidobacteria were resistant to bile concentrations varying from 1 to 3%, and all streptococci were sensitive to bile . Lactase activities were highest for S . thermophilus strains, supporting use of this species in fluid milk and dairy products to aid in the digestion of lactose by consumers . The experimental product evaluated in this study contained 10(7) cfu/ml of both L . Acidophilus and Bifidobacterium spp . and 5 x 10(7) cfu/ml of S . thermophilus . Lactic, but not psychrotrophic, populations were fairly stable during storage . The results suggest that milk formulated with high concentrations of three different genera of probiotic bacteria can be manufactured with commercial strains. J Biochem (Tokyo), 1996 Jun, 119(6), 1076 - 9 Pyridine-promoted factor- and energy-free peptide synthesis systems prepared from various organisms including prokaryote, eukaryote, and mitochondria; Nojima T et al.; We demonstrate here that ribosomes from not only Escherichia coli and Thermus thermophilus {Nitta et al . (1994) J . Biochem . 115, 803-807; ibid., (1995) 118, 841-849} but also yeast and bovine mitochondria catalyze peptide synthesis promoted by a high concentration of pyridine in the absence of soluble protein factors and chemical energy sources, and compare some characteristic features of the reactions among these organisms . Sensitivities against antibiotics, chloramphenicol and cycloheximide, showed the same tendency to those in the in vitro aqueous translation systems of these organisms, suggesting that the basic mechanism for peptide synthesis is the same among these organisms . The optimal concentration of pyridine was centered at 50% for all systems, although the dependencies on the pyridine concentrations and the yields of the products were different from one another . All these systems required Mg2+, and only mitochondrial system showed the extra Mn(2+)-requirement, which enhanced the yield by several fold . The optimum reaction temperatures coincided closely with the growing temperatures of the organisms except for the mitochondrial system, which showed the highest activity above 80 degrees C . The rationale for these observations remains to be solved. Appl Environ Microbiol, 1996 Jun, 62(6), 2191 - 4 Pyrimidine biosynthesis genes (pyrE and pyrF) of an extreme thermophile, Thermus thermophilus; Yamagishi A et al.; We have isolated uracil auxotrophic mutants of an extreme thermophile, Thermus thermophilus . A part of the pyrimidine biosynthetic operon including genes for orotate phosphoribosyltransferase (pyrE) and for orotidine-5'-monophosphate decarboxylase (pyrF) was cloned and sequenced . The pyrE gene can be a bidirectional marker for the gene manipulation system of the thermophile. Appl Environ Microbiol, 1996 Jun, 62(6), 2066 - 73 Analysis of the critical sites for protein thermostabilization by proline substitution in oligo-1,6-glucosidase from Bacillus coagulans ATCC 7050 and the evolutionary consideration of proline residues; Watanabe K et al.; To identify the critical sites for protein thermostabilization by proline substitution, the gene for oligo-1,6- glucosidase from a thermophilic Bacillus coagulans strain, ATCC 7050, was cloned as a 2.4-kb DNA fragment and sequenced . In spite of a big difference in their thermostabilities, B . coagulans oligo-1,6-glucosidase had a large number of points in its primary structure identical to respective points in the same enzymes from a mesophilic Bacillus cereus strain, ATCC 7064 (57%), and an obligately thermophilic Bacillus thermoglucosidasius strain, KP1006 (59%) . The number of prolines (19 for B . cereus oligo-1,6-glucosidase, 24 for B . coagulans enzyme, and 32 for B . thermoglucosidasius enzyme) was observed to increase with the rise in thermostabilities of the oligo-1,6-glucosidases . Classification of proline residues in light of the amino acid sequence alignment and the protein structure revealed by X-ray crystallographic analysis also supported this tendency . Judging from proline residues occurring in B . coagulans oligo-1,6-glucosidase and the structural requirement for proline substitution (second site of the beta turn and first turn of the alpha helix) (K . Watanabe, T . Masuda, H . Ohashi, H . Mihara, and Y . Suzuki, Eur . J . Biochem . 226:277-283, 1994), the critical sites for thermostabilization were found to be Lys-121, Glu-290, Lys-457, and Glu-487 in B . cereus oligo-1,6-glucosidase . With regard to protein evolution, the oligo-1,6-glucosidases very likely follow the neutral theory . The adaptive mutations of the oligo-1,6-glucosidases that appear to increase thermostability are consistent with the substitution of proline residues for neutrally occurring residues . It is concluded that proline substitution is an important factor for the selection of thermostability in oligo-1,6-glucosidases. Curr Opin Biotechnol, 1996 Jun, 7(3), 337 - 42 Biochemistry and genetics of microbial xylanases; Jeffries TW; Xylanases are classified into two major families (10 or F and 11 or G) of glycosyl hydrolases . Both use ion pair catalytic mechanisms and both retain anomeric configuration following hydrolysis . Family 10 xylanases are larger, more complex and produce smaller oligosaccharides; Family 11 xylanases are more specific for xylan . Alkaline-active and extreme-thermophilic enzymes are of particular interest . Such xylanases are being commercialized for bleaching pulps and other applications. Protein Sci, 1996 Jun, 5(6), 1014 - 25 An unusual route to thermostability disclosed by the comparison of Thermus thermophilus and Escherichia coli inorganic pyrophosphatases; Salminen T et al.; The structures of Escherichia coli soluble inorganic pyrophosphatase (E-PPase) and Thermus thermophilus soluble inorganic pyrophosphatase (T-PPase) have been compared to find the basis for the superior thermostability of T-PPase . Both enzymes are D3 hexamers and crystallize in the same space group with very similar cell dimensions . Two rather small changes occur in the T-PPase monomer: a systematic removal of Ser residues and insertion of Arg residues, but only in the C-terminal part of the protein, and more long-range ion pairs from the C-terminal helix to the rest of the molecule . Apart from the first five residues, the three-dimensional structures of E-PPase and T-PPase monomers are very similar . The one striking difference, however, is in the oligomeric interactions . In comparison with an E-PPase monomer, each T-PPase monomer is skewed by about 1 A in the xy plane, is 0.3 A closer to the center of the hexamer in the z direction, and is rotated by approximately 7 degrees about its center of gravity . Consequently, there are a number of additional hydrogen bond and ionic interactions, many of which form an interlocking network that covers all of the oligomeric surfaces . The change can also be seen in local distortions of three small loops involved in the oligomeric interfaces . The complex rigid-body motion has the effect that the hexamer is more tightly packed in T-PPase: the amount of surface area buried upon oligomerization increases by 16% . The change is sufficiently large to account for all of the increased thermostability of T-PPase over E-PPase and further supports the idea that bacterial PPases, most active as hexamers or tetramers, achieve a large measure of their stabilization through oligomerization . Rigid-body motions of entire monomers to produce tighter oligomers may be yet another way in which proteins can be made thermophilic. Genetics, 1996 Jun, 143(2), 811 - 21 Identification, mapping and linkage analysis of randomly amplified DNA polymorphisms in Tetrahymena thermophila; Brickner JH et al.; Using the random amplified polymorphic DNA (RAPD) technique and exploiting the unique genetics of Tetrahymena thermophila, we have identified and characterized 40 DNA polymorphisms occurring between two inbred strains (B and C3) of this ciliated protozoan . These RAPD markers permit the PCR amplification of a DNA species using template DNA from SB1969 (B strain) but fail to do so using DNA from C3-368-5 (C3 strain) . Polymorphisms were mapped to chromosomes using a panel of monosomic strains constructed by crossing B strain-derived nullisomic strains to inbred strain C3 . They map to all five chromosomes and appear to be evenly distributed throughout the genome . Chromosomal groups were then analyzed for linkage using meiotic segregants; four linkage groups were identified in chromosomes 1R 2L, 3 and 5 . The RAPD method appears useful for the construction of a genetic map of the Tetrahymena genome based on DNA polymorphisms. Biochem J, 1996 Jun 1, 316 ( Pt 2), 615 - 22 Thermostable chaperonin from Clostridium thermocellum; Cross SJ et al.; Homologues of the chaperonins Cpn60 and Cpn10 have been purified from the Gram-positive cellulolytic thermophile Clostridium thermocellum . The Cpn60 protein was purified by ATP-affinity chromatography and the Cpn10 protein was purified by gel-filtration, ion-exchange and hydrophobic interaction chromatographies . The identities of the proteins were confirmed by N-terminal sequence analysis and antigenic cross-reactivity . The Cpn60 homologue is a weak, thermostable ATPase (t1/2 at 70 decrees C more than 90 min) with optimum activity (Kcat 0.07 S-1) between 60 degrees C and 70 degrees C . The ATPase activity of the authentic Cpn60 was inhibited by Escherichia coli GroES . The catalytic properties of a recombinant C . thermocellum Cpn60 purified from a GST-Cpn60 fusion protein expressed in E . coli {Ciruela (1995) Ph.D . Thesis, University of Kent} were identical with those of the authentic C . thermocellum Cpn60 . Gel-filtration studies show that at room temperature the Cpn60 migrates mainly as a heptamer . Electron microscopy confirms the presence of complexes showing 7-fold rotational symmetry and also reveals a small number of particles that seem to be tetradecamers with a similar structure to E . coli GroEL complexes. J Bacteriol, 1996 Jun, 178(12), 3654 - 7 Differential domain accessibility to monoclonal antibodies in three different morphological assemblies built up by the S-layer protein of Thermus thermophilus HB8; Caston JR et al.; A collection of 27 monoclonal antibodies (MAbs) against the S-layer protein (P100) of Thermus thermophilus HB8 has been obtained . They have been classified according to their ability to recognize S-layer regions expressed in E . coli from plasmids containing different fragments of its coding gene, slpA . The accessibility of the binding sites in hexagonal, trigonal, or tetragonal assemblies of P100 was analyzed by enzyme-linked immunosorbent assays with six of these MAbs and their respective Fab fragments . When packed hexagonally as the native S-layer (S1 assemblies), only a small region located near the amino terminus of the P1OO was accessible . However, when P1OO was assembled into trigonal (pS2 assemblies) or tetragonal (S2 assemblies) arrays, most of the protein domains analyzed were easily detected, thus suggesting that P1OO is assembled in S2 and pS2 in a similar way and that these two arrangements are quite different from the S1 assembly . Relationships between accessibility and sequence predictions are discussed. J Bacteriol, 1996 Jun, 178(11), 3396 - 8 Differential antibiotic sensitivity determined by the large ribosomal subunit in thermophilic archaea; Ruggero D et al.; Hybrid ribosomes obtained by mixing the ribosomal subunits of the extremely thermophilic archaea Sulfolobus solfataricus and Desulfurococcus mobilis were tested for their sensitivity to selected antibiotics . It is shown that structural differences in the large ribosomal subunits determine qualitatively and quantitatively the patterns of response to alpha-sarcin and paromomycin in these species. J Bacteriol, 1996 Jun, 178(11), 3365 - 8 Purification and characterization of 2-oxoglutarate:ferredoxin oxidoreductase from a thermophilic, obligately chemolithoautotrophic bacterium, Hydrogenobacter thermophilus TK-6; Yoon KS et al.; 2-Oxoglutarate:ferredoxin oxidoreductase from a thermophilic, obligately autotrophic, hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, was purified to homogeneity by precipitation with ammonium sulfate and by fractionation by DEAE-Sepharose CL-6B, polyacrylate-quaternary amine, hydroxyapatite, and Superdex-200 chromatography . The purified enzyme had a molecular mass of about 105 kDa and comprised two subunits (70 kDa and 35 kDa) . The activity of the 2-oxoglutarate:ferredoxin oxidoreductase was detected by the use of 2-oxoglutarate, coenzyme A, and one of several electron acceptors in substrate amounts (ferredoxin isolated from H . thermophilus, flavin adenine dinucleotide, flavin mononucleotide, or methyl viologen) . NAD, NADP, and ferredoxins from Chlorella spp . and Clostridium pasteurianum were ineffective . The enzyme was extremely thermostable; the temperature optimum for 2-oxoglutarate oxidation was above 80 degrees C, and the time for a 50% loss of activity at 70 degrees C under anaerobic conditions was 22 h . The optimum pH for a 2-oxoglutarate oxidation reaction was 7.6 to 7.8 . The apparent Km values for 2-oxoglutarate and coenzyme A at 70 degrees C were 1.42 mM and 80 microM, respectively. J Bacteriol, 1996 Jun, 178(11), 3270 - 4 Characterization of heterologously produced carbonic anhydrase from Methanosarcina thermophila; Alber BE et al.; The gene encoding carbonic anhydrase from Methanosarcina thermophila was hyperexpressed in Escherichia coli, and the heterologously produced enzyme was purified 14-fold to apparent homogeneity . The enzyme purified from E . coli has properties (specific activity, inhibitor sensitivity, and thermostability) similar to those of the authentic enzyme isolated from M . thermophila; however, a discrepancy in molecular mass suggests that the carbonic anhydrase is posttranslationally modified in either E . coli or M . thermophila . Both the authentic and heterologously produced enzymes were stable to heating at 55 degrees C for 15 min but were inactivated at higher temperatures . No esterase activity was detected with p-nitrophenylacetate as the substrate . Plasma emission spectroscopy revealed approximately 0.6 Zn per subunit . As judged from the estimated native molecular mass, the enzyme is either a trimer or a tetramer . Western blot (immunoblot) analysis of cell extract proteins from M . thermophila indicates that the levels of carbonic anhydrase are regulated in response to the growth substrate, with protein levels higher in acetate than in methanol- or trimethylamine-grown cells. Mol Cell Biol, 1996 Jun, 16(6), 2878 - 87 Either of the major H2A genes but not an evolutionarily conserved H2A.F/Z variant of Tetrahymena thermophila can function as the sole H2A gene in the yeast Saccharomyces cerevisiae; Liu X et al.; H2A.F/Z histones are conserved variants that diverged from major H2A proteins early in evolution, suggesting they perform an important function distinct from major H2A proteins . Antisera specific for hv1, the H2A.F/Z variant of the ciliated protozoan Tetrahymena thermophila, cross-react with proteins from Saccharomyces cerevisiae . However, no H2A.F/Z variant has been reported in this budding yeast species . We sought to distinguish among three explanations for these observations: (i) that S . cerevisiae has an undiscovered H2A.F/Z variant, (ii) that the major S . cerevisiae H2A proteins are functionally equivalent to H2A.F/Z variants, or (iii) that the conserved epitope is found on a non-H2A molecule . Repeated attempts to clone an S . cerevisiae hv1 homolog only resulted in the cloning of the known H2A genes yHTA1 and yHTA2 . To test for functional relatedness, we attempted to rescue strains lacking the yeast H2A genes with either the Tetrahymena major H2A genes (tHTA1 or tHTA2) or the gene (tHTA3) encoding hv1 . Although they differ considerably in sequence from the yeast H2A genes, the major Tetrahymena H2A genes can provide the essential functions of H2A in yeast cells, the first such case of trans-species complementation of histone function . The Tetrahymena H2A genes confer a cold-sensitive phenotype . Although expressed at high levels and transported to the nucleus, hv1 cannot replace yeast H2A proteins . Proteins from S . cerevisiae strains lacking yeast H2A genes fail to cross-react with anti-hv1 antibodies . These studies make it likely that S . cerevisiae differs from most other eukaryotes in that it does not have an H2A.F/Z homolog . A hypothesis is presented relating the absence of H2A.F/Z in S . cerevisiae to its function in other organisms. Biochim Biophys Acta, 1996 May 31, 1301(1-2), 105 - 14 Thermoalkalophilic lipase of Bacillus thermocatenulatus . I . molecular cloning, nucleotide sequence, purification and some properties; Schmidt-Dannert C et al.; An expression library was generated by partial Sau3A digestion of genomic DNA from the thermophile Bacillus thermocatenulatus and cloning of DNA fragments in pUC18 in Escherichia coli DH5alpha . Screening for lipase activity identified a 4.5 kb insert in pUC18 which directed the production of lipase in E . coli DH5alpha . A subclone with a 2.2 kb insert was sequenced . The lipase gene codes for a mature lipase of 388 amino acid residues, corresponding to a molecular weight of 43 kDa . As in other Bacillus lipases, an Ala replaces the first Gly in the conserved pentapeptide Gly-X-Ser-X-Gly found in most lipases . The region upstream of the lipase gene contains a Bacillus promoter which directs the expression of lipase in E . coli DH5alpha . The expressed lipase was isolated and purified 312-fold to homogeneity . N-terminal sequencing of the purified lipase revealed a correct cleavage of the preprotein in E . coli DH5alpha . Maximum activity was found at pH 8.0-9.0 with tributyrin and olive oil as substrates and at 60-70 degrees C with p-NPP and olive oil as substrates . The lipase showed high stability at pH 9.0-11.0 and towards various detergents and organic solvents. Proc Natl Acad Sci U S A, 1996 May 28, 93(11), 5281 - 5 Cloning of thermostable DNA polymerases from hyperthermophilic marine Archaea with emphasis on Thermococcus sp . 9 degrees N-7 and mutations affecting 3'-5' exonuclease activity; Southworth MW et al.; Five extremely thermophilic Archaea from hydrothermal vents were isolated, and their DNA polymerases were cloned and expressed in Escherichia coli . Protein splicing elements (inteins) are present in many archaeal DNA polymerases, but only the DNA polymerase from strain GB-C contained an intein . Of the five cloned DNA polymerases, the Thermococcus sp . 9 degrees N-7 DNA polymerase was chosen for biochemical characterization . Thermococcus sp . 9 degrees N-7 DNA polymerase exhibited temperature-sensitive strand displacement activity and apparent Km values for DNA and dNTP similar to those of Thermococcus litoralis DNA polymerase . Six substitutions in the 3'-5' exonuclease motif I were constructed in an attempt to reduce the 3'-5' exonuclease activity of Thermococcus sp . 9 degrees N-7 DNA polymerase . Five mutants resulted in no detectable 3'-5' exonuclease activity, while one mutant (Glul43Asp) had <1% of wild-type activity. Gene, 1996 May 24, 171(1), 103 - 6 Cloning, sequencing and expression of the uvrA gene from an extremely thermophilic bacterium, Thermus thermophilus HB8; Yamamoto N et al.; One of the most important DNA repair systems is the nucleotide (nt) excision repair system . The uvr A gene, which plays an essential role in the prokaryotic excision repair system, was cloned from an extremely thermophilic eubacterium, Thermus thermophilus (Tt) HB8, and its nt sequence was determined . In the amino acid (aa) sequence of Tt UvrA, a characteristic duplicated structure, two nt-binding consensus sequences (Walker's A-type motif) and two zinc finger DNA-binding motifs were found . The aa sequence showed 73% homology with that of Escherichia coli (Ec) . These features suggest that Tt has the same excision repair system as Ec . Upon comparison of the Tt and Ec UvrA, some characteristic aa substitutions were found . The numbers of Arg and Pro residues were increased (from 66 to 81 and from 47 to 55, respectively), and the numbers of Asn and Met residues were decreased (from 33 to 18 and from 18 to 11, respectively) in Tt . The Tt uvr A gene was expressed in Ec under control of the lac promoter . Purified UvrA was stable up to 80 degrees C (at neutral pH) and at pH 2-11 (at 25 degrees C). J Biol Chem, 1996 May 24, 271(21), 12457 - 62 Bacillus stearothermophilus qcr operon encoding rieske FeS protein, cytochrome b6, and a novel-type cytochrome c1 of quinol-cytochrome c reductase; Sone N et al.; The gcr of Bacillus stearothermophilus K1041 encoding three subunits of the quinol-cytochrome c oxidoreductase (cytochrome reductase, b6c1 complex) was cloned and sequenced . The gene (qcrA) for a Rieske FeS protein of 19,144 Da with 169 amino acid residues, and the gene (qcrC) for cytochrome c1 of 27,342 Da with 250 amino acid residues were found at adjacent upstream and downstream sides of the previously reported qcrB (petB) for cytochrome b6 of subunit 25,425 Da with 224 residues (Sone, N., Sawa, G., Sone, T., and Noguchi, S . (1995) J . Biol . Chem . 270, 10612-10617) . The three structural genes for thermophilic Bacillus cytochrome reductase form a transcriptional unit . In the deduced amino acid sequence for the FeS protein, the domain including four cysteines and two histidines binding the 2Fe-2S cluster was conserved . Its N-terminal part more closely resembled the cyanobacteria-plastid type than the proteobacteria-mitochondria type when their sequences were compared . The amino acid sequence of cytochrome c1 was not similar to either type; the thermophilic Bacillus cytochrome c1 is composed of an N-terminal part corresponding to subunit IV with three membrane-spanning segments, and a C-terminal part of cytochrome c reminiscent of cytochrome c-551 of thermophilic Bacillus . The subunit IV in the enzyme of cyanobacteria and plastids is the counterpart of C-terminal part of cytochrome b of proteobacteria and mitochondria . These characteristics indicate that Bacillus cytochrome b6c1 complex is unique. Biochim Biophys Acta, 1996 May 23, 1294(2), 106 - 10 Cloning, characterization and functional analysis of groEL-like gene from thermophilic cyanobacterium Synechococcus vulcanus, which does not form an operon with groES; Furuki M et al.; A gene encoding 57 102 Da polypeptide homologous to groEL of Escherichia coli but accompanying no groES, has been cloned and sequenced from a thermophilic cyanobacterium, Synechococcus vulcanus . The amount of the gene transcript increased several folds by heat shock . The gene was expressed as a minor component of two types of HSP60, and designated as groEL2 . Although expressed and induced well upon heat shock treatment in the E . coli, introduction of the cloned groEL2 gene of S . vulcanus into an E . coli groEL-less mutant did not result in the complementation of heat sensitivity. Arch Microbiol, 1996 May 22, 165(5), 342 - 5 Thermocryptoxanthins: novel intermediates in the carotenoid biosynthetic pathway of Thermus thermophilus Yokoyama A, Shizuri Y, Hoshino T, Sandmann G. Various thermozeaxanthins are the end products of the carotenoid biosynthetic pathway of the thermophilic eubacterium Thermus thermophilus . These compounds are zeaxanthin glucoside esters . Carotenoid analysis and inhibitory studies led to the identification of most of the intermediates of the pathway: beta-carotene, beta-cryptoxanthin, zeaxanthin, and several new carotenoids . The intermediates, identified by various spectroscopic methods as beta-cryptoxanthin glucoside esters carrying fatty acid moieties of different chain lengths, were designated as thermocryptoxanthins . The use of the inhibitors diphenylamine and 2-(4-chlorophenylthio)-triethylamine-HCl resulted in the accumulation of the intermediates phytoene, lycopene, and gamma-carotene derivatives, which normally are present in amounts below the detection limit . The levels of non-esterified glycosides were extremely low . The results presented were used to establish the complete carotenoid biosynthetic pathway of T . thermophilus. FEBS Lett, 1996 May 20, 386(2-3), 260 - 2 5S rRNA binding ribosomal proteins from Thermus thermophilus: identification and some structural properties; Gongadze G et al.; An unusual acidic ribosomal protein from Thermus thermophilus, TL5, that binds to 5S rRNA specifically and strongly, has been investigated . The N-terminal sequence of TL5 does not reveal any homology with known ribosomal proteins . Two large tryptic fragments of TL5 have been isolated and characterized . 5S rRNA protected TL5 and its unstable N-terminal fragment against trypsin action . The 5S rRNA binding ability of TL5 is probably inherent in its N-terminal part . The other 5S rRNA binding ribosomal protein from T . thermophilus, TL4, has been identified as a homolog of the ribosomal protein L5 from Escherichia coli. FEBS Lett, 1996 May 20, 386(2-3), 215 - 8 The cloning, expression and crystallisation of a thermostable arginase; Bewley MC et al.; The gene for the thermostable arginase from the thermophilic bacterium 'Bacillus caldovelox' has been cloned and sequenced . Expression of recombinant arginase at high levels has been achieved in E . coli using an inducible T7 RNA polymerase-based system . A facile purification procedure incorporating a heat-treatment step yielded 0.2 g of recombinant arginase per litre of induced culture . The kinetic properties of the purified recombinant protein are essentially identical to the native enzyme . The recombinant protein has been crystallised and one crystal form is isomorphous to crystals of the native protein. EMBO J, 1996 May 15, 15(10), 2323 - 30 A left-hand beta-helix revealed by the crystal structure of a carbonic anhydrase from the archaeon Methanosarcina thermophila; Kisker C et al.; A carbonic anhydrase from the thermophilic archaeon Methanosarcina thermophila that exhibits no significant sequence similarity to known carbonic anhydrases has recently been characterized . Here we present the structure of this enzyme, which adopts a left-handed parallel beta-helix fold . This fold is of particular interest since it contains only left-handed crossover connections between the parallel beta-strands, which so far have been observed very infrequently . The active form of the enzyme is a trimer with three zinc-containing active sites, each located at the interface between two monomers . While the arrangement of active site groups differs between this enzyme and the carbonic anhydrases from higher vertebrates, there are structural similarities in the zinc coordination environment, suggestive of convergent evolution dictated by the chemical requirements for catalysis of the same reaction . Based on sequence similarities, the structure of this enzyme is the prototype of a new class of carbonic anhydrases with representatives in all three phylogenetic domains of life. Nucleic Acids Res, 1996 May 15, 24(10), 1943 - 9 Programmed DNA rearrangement from an intron during nuclear development in Tetrahymena thermophila: molecular analysis and identification of potential cis-acting sequences; Li J et al.; During macronuclear development in the ciliate Tetrahymena thermophila, extensive rearrangement events occur as DNA deletions . We have studied a developmentally programmed deletion called mse2.9 that occurs within an intron in a gene in both genomic DNA and in an rDNA vector introduced into the cell by transformation . Extensive microheterogeneity at the deletion junctions has been found in caryonidal strains and in the rDNA in transformed cells . A transformation assay has been used to identify sequences required for proper processing of mse2.9 . Models to explain deletion site selection as well as microheterogeneity at junction sites are presented. FEMS Microbiol Lett, 1996 May 15, 139(1), 27 - 35 A modular xylanase from mesophilic Cellulomonas fimi contains the same cellulose-binding and thermostabilizing domains as xylanases from thermophilic bacteria; Clarke JH et al.; The xynC gene from mesophilic Cellulomonas fimi encodes a large 125 kDa modular xylanase (XYLC), consisting of six distinct functional domains . In addition to a single Family 10 catalytic domain, XYLC contains a domain homologous with the nodulation protein, NodB, from nitrogen-fixing bacteria and thermostabilizing and cellulose-binding domains found previously only in xylanases from thermophilic bacteria. Biochem J, 1996 May 15, 316 ( Pt 1), 115 - 22 Cloning and expression of the gene encoding the Thermoanaerobacter ethanolicus 39E secondary-alcohol dehydrogenase and biochemical characterization of the enzyme; Burdette DS et al.; The adhB gene encoding Thermoanaerobacter ethanolicus 39E secondary-alcohol dehydrogenase (S-ADH) was cloned, sequenced and expressed in Escherichia coli . The 1056 bp gene encodes a homotetrameric recombinant enzyme consisting of 37.7 kDa subunits . The purified recombinant enzyme is optimally active above 90 degrees C with a half-life of approx . 1.7 h at 90 degrees C . An NADP(H)-dependent enzyme, the recombinant S-ADH has 1400-fold greater catalytic efficiency in propan-2-ol oxidation than in ethanol oxidation . The enzyme was inactivated by chemical modification with dithionitrobenzoate (DTNB) and diethylpyrocarbonate, indicating that Cys and His residues are involved in catalysis . Zinc was the only metal enhancing S-ADH reactivation after DTNB modification, implicating the involvement of bound zinc in catalysis . Arrhenius plots for the oxidation of propan-2-ol by the native and recombinant S-ADHs were linear from 25 to 90 degrees C when the enzymes were incubated at 55 degrees C before assay . Discontinuities in the Arrhenius plots for propan-2-ol and ethanol oxidations were observed, however, when the enzymes were preincubated at 0 or 25 degrees C . The observed Arrhenius discontinuity therefore resulted from a temperature-dependent, catalytically significant S-ADH structural change . Hydrophobic cluster analysis comparisons of both mesophilic and thermophilic S-ADH and primary- versus S-ADH amino acid sequences were performed . These comparisons predicted that specific proline residues might contribute to S-ADH thermostability and thermophilicity, and that the catalytic Zn ligands are different in primary-alcohol dehydrogenases (two Cys and a His) and S-ADHs (Cys, His, and Asp). Biochemistry, 1996 May 14, 35(19), 6150 - 6 Purification and reconstitution of the glutamate carrier GltT of the thermophilic bacterium Bacillus stearothermophilus; Gaillard I et al.; An affinity tag consisting of six adjacent histidine residues followed by an enterokinase cleavage site was genetically engineered at the N-terminus of the glutamate transport protein GltT of the thermophilic bacterium Bacillus stearothermophilus . The fusion protein was expressed in Escherichia coli and shown to transport glutamate . The highest levels of expression were observed in E . coli strain DH5 alpha grown on rich medium . The protein could be purified in a single step by Ni2+-NTA affinity chromatography after solubilization of the cytoplasmic membranes with the detergent Triton X100 . Purified GltT was reconstituted in an active state in liposomes prepared from E . coli phospholipids . The protein was reconstituted in detergent-treated preformed liposomes, followed by removal of the detergent with polystyrene beads . Active reconstitution was realized with a wide range of Triton X100 concentrations . Neither the presence of glycerol, phospholipids, nor substrates of the transporter was necessary during the purification and reconstitution procedure to keep the enzyme in an active state . In B . stearothermophilus, GltT translocates glutamate in symport with protons or sodium ions . In membrane vesicles derived from E . coli cells expressing GltT, the Na+ ion dependency seems to be lost {Tolner, B., Ubbink-Kok, T., Poolman, B., & Konings, W . N . (1995) Mol . Microbiol . 18, 123-133}, suggesting a role for the lipid environment in the cation specificity . In agreement with the last observation, glutamate transport catalyzed by purified GltT reconstituted in E . coli phospholipid is driven by an electrochemical gradient of H+ but not of Na+. Ned Tijdschr Geneeskd, 1996 May 11, 140(19), 1022 - 5 {Fungi and yeasts isolated in mycological studies in skin and nail infections in The Netherlands, 1992-1993}; Kuijpers AF et al.; OBJECTIVE . To describe fungi and yeasts isolated from skin and nail infections in the Netherlands . DESIGN: Retrospective . SETTING . Centraalbureau voor Schimmelcultures (CBS), Baarn, the Netherlands . METHOD . Results of mycological investigation of skin and nail samples in the period 1992-1993 were analysed . After a clinical diagnosis of mycosis, performed by dermatologists and general practitioners, material was sent to the CBS for mycological research . RESULTS . The clinical diagnosis of onychomycosis was rather accurate, especially if made performed by dermatologists . Mycoses of the skin were sometimes confused with other skin diseases . When microscopical observation showed a positive result, 93% of the cultures were positive as well . The main agent of onychomycosis was Trichophyton rubrum; T . mentagrophytes was more frequently isolated from tinea manuum/pedis and T . tonsurans from tinea corporis/cruris . Epidermophyton floccosum was only isolated from skin lesions and Microsporum canis, T . soudanense and T . verrucosum only from tinea corporis/cruris . The most important yeasts isolated were Trichosporon mucoides, Candida guilliermondii, C . parapsilosis, C . famata and Malassezia furfur . Other fungi isolated were either pigmented (melanin, carotene), thermophilic or belonged to the order of the Onygenales . CONCLUSION . Mycological research to confirm the clinical diagnosis of a skin mycosis is advisable . Species isolated differed in their predilection for different parts of the human body . Yeasts were mainly isolated as double infections . Apart from the dermatophytes there is a special group of fungi which can cause mycoses. Immunol Invest, 1996 May, 25(3), 177 - 83 Immunological detection of lipopolysaccharide antigens of thermophilic campylobacters captured on polymyxin-coated polyester cloth; Gomi K et al.; Cholate-extracted lipopolysaccharide (LPS) antigens from thermophilic campylobacters were captured on polymyxin-coated polyester cloth . The captured antigens were detected by sequential reactions with rabbit anti-Campylobacter antibody, anti-rabbit IgG peroxidase conjugate and chromogenic peroxidase substrate . A polyclonal rabbit antibody elicited against a single Campylobacter-jejuni strain detected the reference strains of the twenty most frequently isolated thermophilic campylobacters in the Lior serotyping scheme . Moreover, LPS antigens of six C . Jejuni Penner serotypes fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and examined by immunoblotting were recognized by four antisera prepared against homologous and heterologous Penner serotypes . The results suggest the potential application of polymyxin-cloth enzyme immunoassay for rapid detection of thermophilic campylobacters where monoclonal antibodies can be raised to possible common LPS epitopes. J Dairy Res, 1996 May, 63(2), 245 - 56 Detection and localization of peptidases in Lactococcus lactis with monoclonal antibodies; Laan H et al.; Monoclonal antibodies against peptidases of Lactococcus lactis were isolated and characterized: PEPN1-4 against a lysyl aminopeptidase PepN, PEPT1-5 against a tripeptidase PepT and PEPD1-3 against a dipeptidase PepD . These monoclonal antibodies reacted specifically with their respective antigens in crude cell extracts of Lc . lactis subspp . cremoris and lactis . A number of monoclonal antibodies cross reacted with proteins of other (lactic acid) bacteria . PEPT1, 2, 4 and 5 cross reacted weakly with a 35 kDa protein in Lactobacillus delbrueckii, while PEPT1 and PEPT2 reacted with proteins in the cell-free extract of Streptococcus thermophilus and Clostridium fervidus . Of the four isolated monoclonal antibodies against PEPN, only PEPN3 cross reacted weakly with a 90 kDa protein in Escherichia coli cell-free extract, and the other three antibody species against PEPN3 cross reacted with 80 kDa proteins of Lb . casei, Lb . delbrueckii, and Str . bovis, but not of Esch . coli . Of the three monoclonal antibodies against PepD, only PEPD1 and PEPD2 cross reacted with 40 kDa proteins of Lb . casei, Lb . delbrueckii and Str . bovis . All PEPN, PEPD and PEPT antibodies reacted with components in cell-free extracts of eleven different Lc . lactis strains, indicating that the peptidases of these strains were very similar to those of Lc . lactis subsp . cremoris WG2 . However, Lc . lactis subsp . hordniae appeared to differ from the other Lc . lactis subspecies since only PEPT1, 2 and 5 reacted with a protein in the cell-free extract . Immunogold labelling of Lc . lactis WG2 with the isolated monoclonal antibodies revealed that PepN, PepD and PepT were located intracellularly . The intracellular location of these peptidases is discussed in relation to the supply of essential amino acids and peptides. J Biochem (Tokyo), 1996 May, 119(5), 1014 - 8 Conversion of the coenzyme specificity of isocitrate dehydrogenase by module replacement; Yaoi T et al.; The coenzyme specificity of isocitrate dehydrogenase from an extreme thermophilic bacterium was converted from NADP-dependent to NAD-dependent by replacing a "module" involved in the coenzyme binding site . The conversion was not possible with the replacement of a few residues that interact with the coenzyme . In addition, the module-replaced mutant dehydrogenase was as stable as the original, wild type enzyme . The results support a previous hypothesis that a module is a structural and functional unit of a protein. Protein Eng, 1996 May, 9(5), 439 - 45 Cumulative stabilizing effects of hydrophobic interactions on the surface of the neutral protease from Bacillus subtilis; Frigerio F et al.; Using genetically engineered mutants of the neutral protease from Bacillus stearothermophilus (BsteNP), it had been shown that the surface-exposed structural motif constituted by Phe63 embedded in a four amino acid hydrophobic pocket is critical for the thermal stability of the thermophilic neutral proteases from Bacilli . To measure the stabilizing contribution of each hydrophobic interaction taking place between Phe63 and the hydrophobic pocket, we grafted this structural motif in the neutral protease from the mesophile Bacillus subtilis (BsubNP) . This was accomplished by first creating the Thr63-->Phe mutant of BsubNP and then generating a series of mutants in which the four amino acids which in thermolysin surround Phe63 and form the hydrophobic pocket were added one after the other . By analysing the thermal stability of each mutant it was found that the 2 degrees C destabilizing effect of the Thr63-->Phe substitution was completely suppressed by the addition of the four amino acid hydrophobic pocket, each replacement providing a stabilizing contribution of approximately 0.8-1 degrees C . These results are discussed in the light of the peculiar mechanism of thermal inactivation of proteolytic enzymes. J Clin Microbiol, 1996 May, 34(5), 1216 - 9 Restriction fragment length polymorphism of flagellin genes of Campylobacter jejuni and/or C . coli isolates from Egypt; Mohran ZS et al.; The conservation of flagellin genes from thermophilic Campylobacter spp . strains isolated in Egypt was evaluated by a restriction fragment length polymorphism (RFLP) assay . The flaA and flaB genes were amplified from 59 independent clinical isolates and digested with EcoRI and PstI, and the resulting patterns were compared with each other and with previously described RFLP groups . The results indicate that the isolates fell into 14 groups for flaA and 11 groups for flaB, 9 of which have been described, and that considerable genetic variability exists among isolates belonging to the same LIO serogroup . In most cases, the flaB gene displayed the same RFLP pattern as that of the flaA gene of the same strain, although some variability was observed . The data suggest that more variability of flagellin genes exists within the LIO serogroups common to Campylobacter field isolates from Egypt than has previously been reported for North American isolates. Biosci Biotechnol Biochem, 1996 May, 60(5), 840 - 6 Purification and characterization of a thermostable alkaline protease from Thermoactinomyces sp . E79 and the DNA sequence of the encoding gene; Lee JK et al.; A thermophilic Thermoactinomyces sp . E79 producing a highly thermostable alkaline protease was isolated from soil . The protease, produced extracellularly by Thermoactinomyces sp . E79, was purified by DEAE-Sepharose CL-6B and Butyl-Toyopearl 650M column chromatography . The relative molecular mass was estimated to be 31,000 by SDS-polyacrylamide gel electrophoresis . Enzyme activity was inhibited by phenylmethylsulfonyl fluoride, suggesting the enzyme to be a serine protease . The optimum temperature for the enzyme activity was 85 degrees C, and about 50% of the original activity remained after incubation at 90 degrees C for 10 min in the presence of Ca2+ . The optimum pH for the enzyme activity was 11.0 and the enzyme was fairly stable from pH 5.0 to 12.0 . The gene for this thermostable alkaline protease was cloned in Escherichia coli and the expressed intracellular enzyme was activated by heat treatment . Sequence analysis showed an open reading frame of 1,152 base pairs, coding for a polypeptide- of 384 amino acids . The polypeptide was composed of a signal sequence (25 amino acids), a prosequence (81 amino acids), and a mature protein of 278 amino acids . The deduced amino acid sequence of the mature protease had high similarity with thermitase, a serine protease from Thermoactinomyces vulgaris, and the extent of sequence identity was 76%. Z Naturforsch {C}, 1996 May-Jun, 51(5-6), 319 - 28 Comparative immunological detection of lipids and carotenoids on peptides of photosystem I from higher plants and cyanobacteria; Makewicz A et al.; Photosystem I preparations were obtained from wild-type tobacco Nicotiana tabacum var . JWB, three chlorophyll-deficient tobacco mutants: Su/su, Su/su var . Aurea and yellow-green leaf patches of the variegated mutant NC 95, Spinacia oleracea and furthermore from the mesophilic cyanobacterium Synechococcus PCC 7942 and the thermophilic cyanobacterium Synechococcus sp. . Peptides from these preparations were analyzed by SDS polyacrylamide gel electrophoresis and transferred for detection of bound lipids and carotenoids according to the Western blot procedure to nitrocellulose membranes . The PS I preparations from the Nicotiana tabacum species and spinach consist of the core complex and the LHCP I complex, the latter containing, however, traces of the LHCP II polypeptides . The core complex consists of the two core peptides with the apparent molecular mass of 66 kDa each and peptides with molecular masses of 22, 20, 19, 17, 16, 10 and 9 kDa . The LHCP I complex contains 4 subunits with molecular masses of 28, 26, 25 and 24 kDa . The PS I preparations of the two mutants Su/su and Su/su var . Aurea contain as impurities traces of the core peptides (D1/D2) and the two chlorophyll-binding peptides (CP43/CP47) of photosystem II . The PS I preparation from the mesophilic and thermophilic cyanobacterium consists of the two core peptides with the apparent molecular mass of 66 kDa and peptides with molecular masses of 16, 14 and 10 kDa . The peptides of the PS I preparations were characterized by specific PS I, CP I and LHCP I antisera . The antiserum to the PS I complex reacts in the Western blot with the homologous peptides of PS I from higher plants, but only with the CP I complex from the two cyanobacteria . In comparative studies with PS II from higher plants the PS I antiserum reacts with the LHCP II complex as expected . The antiserum to the CP I complex reacts only with the 66 kDa peptides of PS I from all objects . There is no cross reaction with the 66 kDa peptides (heterodimer of the D1/D2 peptides) of PS II . The antiserum to the LHCP I complex reacts only with the four LHCP I peptides of PS I from higher plants and as expected with the LHCP II of PS II: Because cyanobacteria do not have LHCP complexes, there is no reaction with the LHCP I antiserum . By means of polyclonal monospecific antisera to lipids it was shown by Western blot procedure that only two lipid species are bound to PS I peptides . The galactolipid monogalactosyldiglyceride is bound to the CP I complex of the Nicotiana tabacum species, spinach and the two cyanobacteria as well as to the LHCP I complex of the higher plants . The phospholipid phosphatidylglycerol is only associated with the CP I complex of the analyzed higher plants and cyanobacteria . With polyclonal monospecific antisera to carotenoids it was demonstrated that beta-carotene, lutein, neoxanthin and zeaxanthin are associated with the CP I complex of the higher plants and the cyanobacteria analyzed . Violaxanthin is also bound to the CP I complex of the two cyanobacteria, whereas it is bound together with neoxanthin to the LHCP I complex of the higher plants. J Eukaryot Microbiol, 1996 May-Jun, 43(3), 198 - 202 An improved method to obtain high molecular weight DNA from purified micro- and macronuclei of Tetrahymena thermophila; Chau MF et al.; An improved method to obtain high molecular weight DNA from purified macro- and micronuclei of Tetrahymena thermophila is described . Micro- and macronuclear DNA obtained using previously described protocols was degraded and not suitable for the cloning of large (>100 kb) DNA fragments . Based on the data reported here, we propose that DNA degradation is mainly due to nuclease activity; some micronuclear DNA degradation is due to mechanical shearing as a result of extended periods of blending . We have made modifications to reduce nuclease degradation by minimizing cell lysis, by the early addition of EDTA and by increasing the EDTA concentration (23 mM) . To reduce mechanical shearing, cell and nuclear suspensions were blended for shorter periods . High molecular weight micro- and macronuclear DNA was obtained using the new protocol. FEMS Microbiol Rev, 1996 May, 18(2-3), 225 - 36 Viruses, plasmids and other genetic elements of thermophilic and hyperthermophilic Archaea; Zillig W et al.; We review and update the work on genetic elements, e.g., viruses and plasmids (exluding IS elements and transposons) in the kingdom Crenarchaeota (Thermoproteales and Sulfolobales) and the orders Thermococcales and Thermoplasmales in the kingdom Euryarchaeota of the archael domain, including unpublished data from our laboratory . The viruses of Crenarchaeota represent four novel virus families . The Fuselloviridae represented by SSVI of S . shibatae and relatives in other Sulfolobus strains have the form of a tailed spindle . The envelope is highly hydrophobic . The DNA is double-stranded and circular . Members of this group have also been found in Methanococcus and Haloarcula . The Lipothrivciridae (e.g., T TV1 to 3) have the form of flexible filaments . They have a core containing linear double-stranded DNA and DNA-binding proteins which is wrapped into a lipid membrane . The "Bacilloviridae" (e.g., TTV4 and SIRV) are stiff rods lacking this membrane, but also featuring linear double-stranded DNA and DNA-binding proteins . Both virus types carry on both ends structures involved in the attachment to receptors . Both types are represented in Thermoproteus and Sulfolobus . The droplet-formed novel Sulfolobus virus SNDV represents the "Guttaviridae" containing circular double-stranded DNA . Though head and tail viruses distantly resembling T phages or lambdoid phages were seen electronmicroscopically in solfataric water samples, no such virus has so far been isolated . SSV1 is temperate, TTV1 causes lysis after induction, the other viruses found so far exist in carrier states . The hosts of all but TTV1 survive virus production . We discuss the implications of the nature of these viruses for understanding virus evolution . The plasmids found so far range in size from 4.5 kb to about 40 kb . Most of them occur in high copy number, probably due to the way of their detection . Most are cryptic, pNOB8 is conjugative, the widespread pDL10 alleviates in an unknown way autotrophic growth of its host Desulfurolobus by sulfur reduction . The plasmid pTIK4 appears to encode a killer function . pNOB8 has been used as a vector for the transfer of the lac S (beta-galactosidase) gene into a mutant of S . solfataricus. FEMS Microbiol Rev, 1996 May, 18(2-3), 215 - 24 Glyceraldehyde-3-phosphate dehydrogenase from Thermotoga maritima: strategies of protein stabilization; Jaenicke R; The molecular origin of protein stability has been the subject of active research for more than a generation (R . Jaenicke (1991) Eur . J . Biochem . 202, 715-728) . Faced with the discovery of extremophiles, in recent years the problem has gained momentum, especially because of its biotechnological potential . In analyzing a number of enzymes from the hyperthermophilic bacterium Thermotoga maritima, it has become clear that the excess free energy of stabilization is equivalent to only a few weak bonds (delta delta Gstab approximately equal to 50 kJ/mol) . As taken from the comparison of homologous enzymes from mesophiles, thermophiles and hyperthermophiles, these accumulate from local interactions (especially ion pairs), enhanced secondary or supersecondary structure, and improved packing of domains and/or subunits, without significantly altering the overall topology . In this review, glyceraldehyde-3-phosphate dehydrogenase will be discussed as a representative example to illustrate possible adaptive strategies to the extreme thermal stress in hydrothermal vents. Appl Environ Microbiol, 1996 May, 62(5), 1723 - 7 Isolation of Thermus strains from hot composts (60 to 80 degrees C); Beffa T et al.; High numbers (10(7) to 10(10) cells per g {dry weight}) of heterotrophic, gram-negative, rod-shaped, non-sporeforming, aerobic, thermophilic bacteria related to the genus Thermus were isolated from thermogenic composts at temperatures between 65 and 82 degrees C . These bacteria were present in different types of wastes (garden and kitchen wastes and sewage sludge) and in all the industrial composting systems studied (open-air windows, boxes with automated turning and aeration, and closed bioreactors with aeration) . Isolates grew fast on a rich complex medium at temperatures between 40 and 80 degrees C, with optimum growth between 65 and 75 degrees C . Nutritional characteristics, total protein profiles, DNA-DNA hybridization (except strain JT4), and restriction fragment length polymorphism profiles of the DNAs coding for the 16S rRNAs (16S rDNAs) showed that Thermus strains isolated from hot composts were closely related to Thermus thermophilus HB8 . These newly isolated T . thermophilus strains have probably adapted to the conditions in the hot-compost ecosystem . Heterotrophic, ovalspore-forming, thermophilic bacilli were also isolated from hot composts, but none of the iso