Gene, 1989 Apr 30, 77(2), 253 - 63 Genes encoding 5S rRNA and tRNAs in the extremely thermophilic archaebacterium Methanothermus fervidus; Haas ES et al.; Methanothermus fervidus was shown to have two 5S rRNA-encoding genes linked in rRNA operons to 16S and 23S rRNA-encoding genes . Sequencing of a cloned 5S rRNA gene confirmed that M . fervidus is a member of the Methanobacteriales, although its 5S rRNA is also similar in both primary sequence and predicted secondary structure to the 5S rRNA of the non-methanogenic, but also extremely thermophilic archaebacterium, Thermococcus celer . Two clusters of tRNA genes have also been cloned and sequenced form M . fervidus . The smaller cluster, cloned in pET5401, is composed of 5'-tRNA(UGUThr)-tRNA(UGGPro)-tRNA(GUCAsp)-tRNA(UUUL ys)-3' and the larger cluster, cloned in pET5475, is composed of 5'-tRNA(GUUAsn)-tRNA(CAUMet)-tRNA(UUCGlu)-tRNA(UAGL eu)-tRNA(GUGHis)-3' . The encoded tRNAs, with the exception of the tRNA(Leu), translate abundant codons in M . fervidus . The tRNA genes do not contain introns or encode 3'-terminal CCA residues . Homologous clusters of tRNA genes have been sequenced from Methanococcus vannielii and Methanococcus voltae, so that comparisons of transcription signals, gene organizations and primary sequences can be made and features possibly related to thermostability identified . During evolution, a 5S rRNA gene appears to have been incorporated into the cluster of tRNA genes in the methanococci but not in M . fervidus. Nucleic Acids Res, 1989 Apr 25, 17(8), 3247 - 60 Tetrahymena micronuclear sequences that function as telomeres in yeast; Shampay J et al.; We explored the ability of S . cerevisiae to utilize heterologous DNA sequences as telomeres by cloning germline (micronuclear) DNA from Tetrahymena thermophila on a linear yeast plasmid that selects for telomere function . The only Tetrahymena sequences that functioned in this assay were (C4A2)n repeats . Moreover, these repeats did not have to be derived from Tetrahymena telomeres, although we show that micronuclear telomeres (like macronuclear telomeres) of Tetrahymena terminate in (C4A2)n repeats . Chromosome-internal restriction fragments carrying (C4A2)n repeats also stabilized linear plasmids and were elongated by yeast telomeric repeats . In one case, the C4A2 repeat tract was approximately 1.5 kb from the end of the genomic Tetrahymena DNA fragment that was cloned, but this 1.5 kb of DNA was missing from the linear plasmid . Thus, yeast can utilize internally located tracts of telomere-like sequences, after the distal DNA is removed . The data provide an example of broken chromo-some healing, and underscore the importance of the telomeric repeat structure for recognition of functional telomeric DNA in vivo. Gene, 1989 Apr 15, 77(1), 11 - 9 Characterization and cloning of MwoI (GCN7GC), a new type-II restriction-modification system from Methanobacterium wolfei; Lunnen KD et al.; R.MwoI, a type-II restriction enzyme with the new specificity 5'-GCN7GC-3', was found in extracts of the thermophilic archaebacterium, Methanobacterium wolfei . R.MwoI cleaves duplex DNA producing fragments with 3-nt, 3'-terminal extensions, thus: GCN5/N2GC . The genes coding for the MwoI restriction and modification enzymes were cloned into Escherichia coli on the plasmid vector pBR322 . The clones synthesize a low level of R.MwoI endonuclease . The plasmids display incomplete MwoI-specific modification, suggesting that the clones synthesize a low level of the M.MwoI methyltransferase, too. Eur J Biochem, 1989 Apr 15, 181(1), 261 - 8 Evidence for cytochrome oxidase subunit I and a cytochrome c--subunit II fused protein in the cytochrome 'c1aa3' of Thermus thermophilus . How old is cytochrome oxidase? Buse G, Hensel S, Fee JA. The terminal cytochrome c1aa3 of the respiratory chain of Thermus thermophilus has been isolated and purified to homogeneity by a novel procedure . The two subunit proteins (55 and 33 kDa) have been characterized chemically . Computer searches with partial amino acid sequences obtained from both subunits show that the larger subunit belongs to the cytochrome oxidase subunit I protein family while the smaller covalently heme-binding subunit is not a cytochrome c1 but appears to be a fused protein between cytochrome c and cytochrome oxidase subunit II . With respect to the 16-S rRNA-derived phylogeny of procaryotes, the results show that the genetic information for an O2-reacting cytochrome oxidase (EC 1.9.3.1) existed already in early eubacteria. J Biol Chem, 1989 Apr 15, 264(11), 6092 - 6 Steady state kinetics of proton translocation catalyzed by thermophilic F0F1-ATPase reconstituted in planar bilayer membranes; Muneyuki E et al.; The proton-translocating ATPase of the thermophilic bacterium PS3 was reconstituted into planar phospholipid bilayers by the previously reported method (Hirata, H., Ohno, K., Sone, N., Kagawa, Y., and Hamamoto, T . (1986) J . Biol . Chem . 261, 9839-9843), and the relationship between the electric current induced by ATP and the concentration of ATP was examined . The magnitude of the electric current generated upon addition of ATP followed simple Michaelis-Menten type kinetics, and the Michaelis constant was found to be 0.14 mM under our conditions . This value is close to the values reported for F1- or F0F1-ATPase in its steady state catalytic cycle, indicating that the proton translocation is coupled to the steady state ATPase reaction . The relationship between the Km value and the membrane potential was also examined under the voltage-clamped condition, and we found that there was no apparent dependence of the Km on membrane voltage . These results together with the previous data suggest that the voltage dependence residues in some step that defines the apparent Vmax rather than Km in the reaction cycle, and proton translocation is not directly coupled to this ATP binding step. J Mol Biol, 1989 Apr 5, 206(3), 411 - 24 Genomic organization of the glyceraldehyde-3-phosphate dehydrogenase gene family of Caenorhabditis elegans; Huang XY et al.; Glyceraldehyde-3-phosphate dehydrogenase (GAPDHase) is encoded by four genes designated gpd-1 through gpd-4 in the nematode Caenorhabditis elegans . gpd-1 has been isolated and sequenced, and is shown here to have a nearly identical copy (gpd-4) with respect to coding and regulatory flanking sequence information as well as to the placement of its two introns . Both genes, which are separated by 250,000 to 300,000 base-pairs were assigned to chromosome II by in situ hybridization and physically linked to a DNA polymorphism located near unc-4 on the genetic map . The genes gpd-2 and gpd-3 are also nearly identical with each other but differ from the gpd-1 and gpd-4 pair with respect to the positions of their two introns and a cluster of amino acid changes within the amino-terminal region of the enzyme . Furthermore, one gene from each pair (gpd-4 and gpd-2) exhibits a single amino acid substitution at positions heretofore known to be conserved in all other systems so far examined including the extreme thermophiles . gpd-2 and gpd-3 are organized as a direct tandem repeat separated by only 244 base-pairs . They have been assigned to an 85,200 base-pair contig that maps to the left end of the X chromosome . The absence of gpd-3 from C . elegans var . Bergerac was used as a marker to map the gpd-2,3 gene pair near unc-20 . Northern analyses have shown that gpd-1 and gpd-4 are preferentially expressed in embryos, while the expression of gpd-2 and gpd-3 increases during postembryonic development . These analyses indicate that the gpd-1,4 gene pair encodes the minor isoenzyme, GAPDHase-1, present in all cells of the nematode while the other gene pair (gpd-2,3) encodes the major isoenzyme, GAPDHase-2, preferentially expressed in the bodywall muscle . The G + T-rich and T-rich regions essential for vertebrate beta-globin polyadenylation were also observed for gpd-3. Exp Cell Res, 1989 Apr, 181(2), 574 - 8 Concanavalin A inhibits mating type recognition in Tetrahymena; Pagliaro L et al.; The lectin concanavalin A (Con A) inhibits adhesion of cells of complementary mating types into pairs during conjugation in the ciliate Tetrahymena thermophila . Distinct changes in protein synthesis occur in conjugating Tetrahymena, starting before cells have paired, as a result of a preliminary interaction, costimulation, involving nonadhesive, contact-mediated, specific cell-cell recognition . We report here that ConA inhibits costimulation-induced protein synthesis changes . We interpret this result as evidence that Con A inhibits cellular recognition, independent of cell-cell adhesion, in Tetrahymena. FEMS Microbiol Lett, 1989 Apr, 49(2-3), 145 - 50 Identification of 'Campylobacter upsaliensis' and other catalase-negative campylobacters from paediatric blood cultures by numerical analysis of electrophoretic protein patterns; Owen RJ et al.; Twenty-one strains of catalase-negative campylobacters from paediatric blood cultures were characterized by one-dimensional SDS-PAGE of cellular proteins . A further 11 Campylobacter strains were included for reference purposes . The partial protein patterns were used as the basis of a numerical analysis, which showed that 17 hippurate-negative strains had a high similarity to 'C . upsaliensis' (r greater than or equal to 0.82) irrespective of their geographical location, and that three hippurate-positive isolates had a high similarity (r greater than or equal to 0.87) to C . jejuni subsp . jejuni . One hippurate-positive CNW strain was not identified . The analysis of SDS-PAGE protein patterns proved an excellent method of characterizing these thermophilic campylobacter as they were difficult to identify by traditional methods. J Clin Microbiol, 1989 Apr, 27(4), 668 - 70 Direct isolation of atypical thermophilic Campylobacter species from human feces on selective agar medium; Walmsley SL et al.; Campylobacter upsaliensis is the name which has been proposed for a new group of thermophilic campylobacter strains which differ from C . jejuni and C . coli in having a negative or weak catalase reaction . Primary isolation of these strains from human feces has been achieved only by use of filtration techniques . We report here direct isolation of strains corresponding to C . upsaliensis from stools of six children . The strains were isolated on a newly described campylobacter-selective medium . The strains were oxidase positive, hippurate negative, nitrate positive, negative for H2S in triple sugar iron, and susceptible to cephalothin (30-micrograms disk) and nalidixic acid (30-micrograms disk), and they grew at 37 and 43 degrees C, but not at 25 degrees C . The selective medium used was a blood-free, charcoal-based medium consisting of Columbia agar base, activated charcoal, cefoperazone (32 micrograms/ml), vancomycin (20 micrograms/ml), and cycloheximide (100 micrograms/ml) . The medium supported the growth of the weakly reacting or catalase-negative strains, with colony counts equivalent to those obtained on antibiotic-free horse blood agar . These strains could not be isolated directly from stool on Skirrow medium, and colony counts confirmed that this medium could not support a low inoculum of these organisms . The clinical significance of these strains is unknown . We conclude that C . upsaliensis can be isolated directly from stool by using a selective medium, without the need for filtration. Immun Infekt, 1989 Apr, 17(2), 59 - 60 {Farmer's lung: IgG-subclass reactivities against thermophilic actinomycetes}; Reese G et al.; IgG subclass reactivities of patients suffering from farmer's lung, healthy family members and blood donors were tested for specific binding to Micropolyspora faeni extract by means of immunoblot techniques (IEF-Print, Western Blot) . In patients' sera IgG1 and IgG2 had shown the strongest reactivities while IgG3- and IgG4-reactivities were not found in all cases . Strong IgG2-reactivities against acidic proteins (pl 3-5) of M . faeni seem to distinguish between patients and exposed or not exposed controls, respectively. J Bacteriol, 1989 Apr, 171(4), 1788 - 92 Purification and characterization of ATP:citrate lyase from Hydrogenobacter thermophilus TK-6; Ishii M et al.; ATP:citrate lyase {ATP citrate (pro-3S)-lyase; EC 4.1.3.8} was purified and characterized from the cells of Hydrogenobacter thermophilus, an aerobic, thermophilic, hydrogen-oxidizing bacterium which fixes carbon dioxide by a reductive carboxylic acid cycle . The enzyme was quite stable, even in the absence of sulfhydryl reagents . Optimum pH for reaction was 6.7 to 6.9, and optimum temperature was around 80 degrees C . The molecular weight of native enzyme was estimated to be 260,000 by gel filtration analysis, and that of a subunit was estimated to be 43,000 by sodium dodecyl sulfate-polyacrylamide gel analysis . Km values for reaction components were as follows: citrate, 6.25 mM; ATP, 650 microM; coenzyme A, 40.8 microM; and Mg2+, 8 mM . The enzyme showed citrate synthase activity in the presence of Mg2+, but the reaction rate was very low (less than 1/200 of the lyase activity). Acta Crystallogr B, 1989 Apr 1, 45 ( Pt 2), 190 - 9 Cryocrystallography of ribosomal particles; Hope H et al.; Crystals suitable for X-ray study have been prepared from biochemically active ribosome particles or their complexes with tRNA and polypeptide chains . At ambient temperature the useful lifetime of these crystals under synchrotron irradiation is limited to a few minutes . However, upon cooling to cryogenic temperatures around 85 K, the original resolution limit (up to 4.5 A) can be recorded and radiation damage is virtually eliminated . Hence it has become possible to collect a complete data set from one single crystal . Crystals were cooled as rapidly as possible, either in a cold gas stream, or by immersion in liquid propane . Before cooling crystals were transferred either to an inert hydrocarbon environment, or to solutions similar to the crystallizing ones but with a higher viscosity . In several cases soaking in a cryosolvent was required . Crystallographic data were collected with intense synchrotron radiation . Full data sets have been measured for native and derivatized crystals of 50S ribosomal subunits from H . marismortui as well as from their complexes with tRNA and nascent polypeptide chains, from the wild type and a mutant of 50S subunits from B . stearothermophilus, and from crystals of native and derivatized 30S ribosomal subunits from T . thermophilus. Lab Anim, 1989 Apr, 23(2), 126 - 32 Campylobacter jejuni infection within a laboratory animal production unit; Meanger JD et al.; A conventional laboratory animal production unit in which rats, mice, guineapigs and rabbits were bred in one building and cats maintained in a separate, but adjacent area was examined for the presence of intestinal thermophilic Campylobacter spp . Campylobacter jejuni was recovered from 18.84% of 552 animals . The infection rate was highest amongst the cats (51.7%), with rats being the second most commonly infected (23.2%), whereas only 7.7% of guineapigs and a single rabbit (1%) were positive . Campylobacter-like organisms were cultured from 10% of the mice, but these bacteria failed to grow on subsequent subculturing . By using bacterial restriction endonuclease DNA analysis (BRENDA), a single type of C . jejuni was identified from all isolates recovered from the rats, guineapigs and a rabbit, suggesting a common source of infection . In contrast, there were 5 different BRENDA patterns derived from cat isolates . No isolates of C . jejuni were obtained from humans working within the unit or from animal bedding or the immediate environment, although it was suggested that the organism may have entered and spread within the unit from sawdust. Mol Microbiol, 1989 Apr, 3(4), 541 - 51 Primary structure, functional organization and expression of nitrogenase structural genes of the thermophilic archaebacterium Methanococcus thermolithotrophicus; Souillard N et al.; Two regions of homology to Anabaena nifH (nitrogenase Fe protein) were detected in the total DNA of the thermophilic nitrogen-fixing archaebacterium Methanococcus thermolithotrophicus . A 2.8 kb HindIII fragment carrying one of these regions was previously cloned and shown to contain a nifH gene (Souillard et al., 1988) now referred to as ORFnifH2 . A 3.4 kb PstI fragment and an overlapping 3.8 kb BglII fragment, containing the second region of homology, were cloned, and a DNA region of 4073 bp was sequenced . It contained four complete open reading frames (ORFs) (ORF nifH1, ORF105, ORF128, ORFnifD) and two truncated ORFs (ORFnifK and ORF96) . Five ORFs were transcribed in the same direction in the order of ORFnifH1-ORF105-ORF128-ORFnifD-ORFnifk . ORFnifH1, ORFnifD and ORFnifK were assigned from their similarity to eubacterial nifH and nifDK (nitrogenase MoFe protein) genes . Transcription studies showed that ORFnifH1 and ORFnifD were expressed only under nitrogen-fixation conditions, whereas no ORFnifH2 mRNA was detected under the same conditions . A DNA probe containing ORFnifH1 hybridized with a 1.8 kb mRNA, as detected by a Northern blotting experiment . A transcriptional start site was localized 87 and 88 bp upstream from the ATG codon of ORFnifH1 . This site is preceded, 21 bp upstream, by the sequence 5'-TTTATATA-3' already found at the same position in several archaebacterial promoters . ORFnifH1 mRNA was too small to encode ORFnifDK . This was confirmed by the fact that another transcription start site was localized 85 bp upstream from the ATG codon of ORFnifD. Biochimie, 1989 Apr, 71(4), 559 - 63 Thermostable alanine dehydrogenase of Bacillus sp . DSM730: gene cloning, purification, and characterization; Nagata S et al.; We have cloned the thermostable alanine dehydrogenase (EC 1.4.1.1) gene from a thermophile, Bacillus sp . DSM730, into Escherichia coli C600 with a vector plasmid, pBR322 . The enzyme was overproduced by the transformed cells, and purified to homogeneity with a yield of 69% by heat treatment and another step . The enzyme has a molecular weight of about 250,000 and consists of 6 subunits identical in molecular weight (43,000) . It is not inactivated by heat treatment at 75 degrees C for 60 min, or incubation in the pH range of 5.5-10.5 at 55 degrees C for 10 min . The enzyme ctalyzes the oxidative deamination of L-serine in addition to L-alanine . The oxo analogue of serine is as reactive as pyruvate . Thus, the enzyme differs markedly from alanine dehydrogenases so far studied. Mol Gen Genet, 1989 Apr, 216(2-3), 334 - 9 Gene sequence for the 9 kDa component of Photosystem II from the cyanobacterium Phormidium laminosum indicates similarities between cyanobacterial and other leader sequences; Wallace TP et al.; A 9 kDa polypeptide which is loosely attached to the inner surface of the thylakoid membrane and is important for the oxygen-evolving activity of Photosystem II in the thermophilic cyanobacterium Phormidium laminosum has been purified, a partial amino acid sequence obtained and its gene cloned and sequenced . The derived amino acid sequence indicates that the 9 kDa polypeptide is initially synthesised with an N-terminal leader sequence of 44 amino acids to direct it across the thylakoid membrane . The leader sequence consists of a positively charged N-terminal region, a long hydrophobic region and a typical cleavage site . These features have analogous counterparts in the "thylakoid-transfer domain" of lumenal polypeptides from chloroplasts of higher plants . These findings support the view of the proposed function of this domain in the two-stage processing model for import of lumenal, nuclear-encoded polypeptides . In addition, there is striking primary sequence homology between the leader sequences of the 9 kDa polypeptide and those of alkaline phosphatase (from the periplasmic space of Escherichia coli) and, particularly in the region of the cleavage site, the 16 kDa polypeptide of the oxygen-evolving apparatus in the thylakoid lumen of spinach chloroplasts. Zentralbl Bakteriol Mikrobiol Hyg {B}, 1989 Apr, 187(4-6), 312 - 23 {The significance of amoebae and other protozoa in water conduit systems in dental units}; Michel R et al.; In a longitudinal study over a period of ten weeks, the water conduit systems of seven dental surgeries with a total of 20 dental units in which legionellae had been detected in an earlier study (3) were investigated as a complement to extended microbiological tests (4) for the occurrence of free-living amebae and other protozoa . In 96% of all water samples, one or several ameba species were detected . These included 14 strains of the genus Naegleria, but no acanthamoebae . The naeglerias isolated were not thermophilic and thus belong to the N . gruberi complex, which does not contain any pathogenic strains . In two water samples, two different species of small freeliving nematodes were demonstrated . The simultaneous investigation of samples from the warm water supply in the individual dental surgeries revealed an ameba contamination in 24.9% of the samples . A nonpathogenic strain of the genus Protacanthamoeba was demonstrated once . The possible role of the trophozoites of various ameba species as hosts for an intracellular proliferation of the legionella strains demonstrated at the same time (cf . 4) is discussed . The occasional protective intracellular inclusion of legionellae in trophozoites of various species and in cysts of acanthamoebae might be an explanation for the resistance of legionellae to disinfection measures which has been repeatedly observed . This observation will have to be taken into consideration in sterilization and disinfection projects. J Mol Biol, 1989 Mar 20, 206(2), 397 - 406 Engineering protein thermal stability . Sequence statistics point to residue substitutions in alpha-helices; Menendez-Arias L et al.; Amino acid sequences have been compared for thermophilic and mesophilic molecules from six different protein families, which include lactate and glyceraldehyde-3-phosphate dehydrogenases, triose phosphate isomerases, superoxide dismutases, thermolysins and subtilisins . Since a three-dimensional structure was known for at least one of the sequences in each family, analysis of preferred residue substitutions, presumably to achieve thermal stability, could be examined from a structural context . The overall results, which are generally consistent across all the families, suggested decreased flexibility and increased hydrophobicity in alpha-helical regions as the main stabilizing principles . The most favoured residual exchanges, hopefully useful in engineering stability into proteins, are discussed. FEBS Lett, 1989 Mar 13, 245(1-2), 253 - 60 The complete amino acid sequence of ribosomal protein S18 from the moderate thermophile Bacillus stearothermophilus; McDougall J et al.; The amino acid sequence of ribosomal protein S18 from Bacillus stearothermophilus has been completely determined by automated sequence analysis of the intact protein as well as of peptides derived from digestion with Staphylococcus aureus protease at pH 4.0 and cleavage with cyanogen bromide . The carboxy-terminal region was verified by both amino acid analyses of chymotryptic peptides and by mass spectrometry from the terminal region . The protein contains 77 amino acid residues and has an Mr of 8838 . Comparison of this sequence with the sequences of the S18 proteins from tobacco and liverwort chloroplasts and E . coli shows a relatively high similarity, ranging from 42 to 55% identical residues with the B . stearothermophilus S18 protein . The regions of homology common to all four proteins consist of several positively charged sections spanning the entire length of the protein. Appl Environ Microbiol, 1989 Mar, 55(3), 722 - 32 Effect of thermal additions on the density and distribution of thermophilic amoebae and pathogenic Naegleria fowleri in a newly created cooling lake; Tyndall RL et al.; Pathogenic Naegleria fowleri is the causative agent of fatal human amoebic meningoencephalitis . The protozoan is ubiquitous in nature, and its presence is enhanced by thermal additions . In this investigation, water and sediments from a newly created cooling lake were quantitatively analyzed for the presence of thermophilic amoebae, thermophilic Naegleria spp., and the pathogen Naegleria fowleri . During periods of thermal additions, the concentrations of thermophilic amoebae and thermophilic Naegleria spp . increased as much as 5 orders of magnitude, and the concentration of the pathogen N . fowleri increased as much as 2 orders of magnitude . Concentrations of amoebae returned to prior thermal perturbation levels within 30 to 60 days after cessation of thermal additions . Increases in the thermophilic amoeba concentrations were noted in Savannah River oxbows downriver from the Savannah River plant discharge streams as compared with oxbows upriver from the discharges . Concentrations of thermophilic amoebae and thermophilic Naegleria spp . correlated significantly with temperature and conductivity . Air samples taken proximal to the lake during periods of thermal addition showed no evidence of thermophilic Naegleria spp . Isoenzyme patterns of the N . fowleri isolated from the cooling lake were identical to patterns of N . fowleri isolated from other sites in the United States and Belgium. J Biochem (Tokyo), 1989 Mar, 105(3), 362 - 6 Molecular cloning and the nucleotide sequence of the Clostridium thermocellum trpE gene; Sato S et al.; The trpE gene of Clostridium thermocellum, a moderately thermophilic and absolutely anaerobic bacterium, was cloned by its ability to complement growth of an Escherichia coli tryptophan auxotroph (trpE) deficient in anthranilate synthase I . The nucleotide sequence of trpE and its flanking region was determined . The trpE gene overlapped at the termination codon with a putative initiation codon of trpG (trp{G}D), as deduced from the amino acid sequence homology with anthranilate synthase II of Serratia marcescens . S1-nuclease mapping of the trpE transcript produced in E . coli cells suggested that the promoter of C . thermocellum was utilized by E . coli . The amino acid sequence of anthranilate synthase I of C . thermocellum predicted from the nucleotide sequence is more similar to that of an extremely thermophilic bacterium, Thermus thermophilus HB8, than that of mesophilic bacteria. Mol Cell Biol, 1989 Mar, 9(3), 1092 - 9 Accurate processing and amplification of cloned germ line copies of ribosomal DNA injected into developing nuclei of Tetrahymena thermophila; Yao MC et al.; The ciliate Tetrahymena thermophila contains a chromosomally integrated copy of the rRNA genes (rDNA) in its germinal (micronuclear) genome . These genes are excised from the chromosome through a process involving site-specific DNA breakage, become linear palindromic molecules with added telomeres, and are greatly amplified during development of the somatic nucleus (macronucleus) . In this study, we cloned a 15-kilobase segment of the germ line DNA containing these genes and injected it into developing macronuclei of T . thermophila . Up to 11% of injected cells were transformed to the paromomycin-resistant phenotype specified by the injected DNA . Transformation efficiency was dependent on the developmental stages of the injected cells and the integrity of the injected DNA but not the DNA concentration or conformation . The injected DNA was apparently processed and amplified correctly to produce rDNA molecules with the expected linear palindromic structure which carried the appropriate physical markers . Thus, the 15-kilobase DNA contained all cis-acting sequences sufficient for the DNA-processing events leading to rDNA amplification in T . thermophila. Proc Natl Acad Sci U S A, 1989 Mar, 86(6), 1791 - 5 Nucleocytoplasmic transport of ribosomes in a eukaryotic system: is there a facilitated transport process? Khanna-Gupta A, Ware VC. We have examined the kinetics of the process by which ribosomes are exported from the nucleus to the cytoplasm using Xenopus laevis oocytes microinjected into the germinal vesicle with radiolabeled ribosomes or ribosomal subunits from X . laevis, Tetrahymena thermophila, or Escherichia coli . Microinjected eukaryotic mature ribosomes are redistributed into the oocyte cytoplasm by an apparent carrier-mediated transport process that exhibits saturation kinetics as increasing amounts of ribosomes are injected . T . thermophila ribosomes are competent to traverse the Xenopus nuclear envelope, suggesting that the basic mechanism underlying ribosome transport is evolutionarily conserved . Microinjected E . coli ribosomes are not transported in this system, indicating that prokaryotic ribosomes lack the "signals" required for transport . Surprisingly, coinjected small (40S) and large (60S) subunits from T . thermophila are transported significantly faster than individual subunits . These observations support a facilitated transport model for the translocation of ribosomal subunits as separate units across the nuclear envelope whereby the transport rate of 60S or 40S subunits is enhanced by the presence of the partner subunit . Although the basic features of the transport mechanism have been preserved through evolution, other aspects of the process may be mediated through species-specific interactions . We hypothesize that a species-specific nuclear 40S-60S subunit association may expedite the transport of individual subunits across the nuclear envelope. Development, 1989 Mar, 105(3), 447 - 56 Non-genic inheritance of cellular handedness; Nelsen EM et al.; Ciliates exhibit an asymmetry in arrangement of surface structures around the cell which could be termed handedness . If the usual order of placement of structures defines a 'right-handed' (RH) cell, then a cell with this order reversed would be 'left-handed' (LH) . Such LH forms appear to be produced in Tetrahymena thermophila through aberrant reorganization of homopolar doublets back to the singlet condition . Four clones of LH forms were selected and subjected to genetic analysis to test whether this drastic phenotypic alteration resulted from a nuclear genetic change . The results of this analysis indicate that the change in handedness is not due to a genetic change in either the micronucleus or macronucleus . The LH form can, under certain circumstances, revert to the RH form, but typically it propagates itself across both vegetative and sexual generations with similar fidelity . While this analysis does not formally rule out certain possibilities of nuclear genic control involving regulatory elements transmitted through the cytoplasm, when the circumstances of origin and propagation of the LH condition are taken into account direct cortical perpetuation seems far more likely . Here we outline a conceptual framework centred on the idea of longitudinally propagated positional information; the positive evidence supporting this idea as well as further application of the idea itself are presented in the accompanying paper. J Cell Sci, 1989 Mar, 92 ( Pt 3), 349 - 52 The transient division block in the D9ts mutant of Tetrahymena is inducible at every cell cycle stage; Cleffmann G; The temperature-sensitive mutant D9 of Tetrahymena thermophila doubles its size at restrictive temperature . It does so by complete cessation of cell division for a limited time . After resumption of proliferation, division rate and specific growth rate are the same as at the permissive temperature, thereby maintaining the new cell size . In this study a detailed analysis of the process of controlling the new cell size is presented, by probing the temperature sensitivity of cell cycle phases . It will be shown that high temperature affects the size-controlling system immediately upon shift in temperature . Temperature pulses are effective at every stage in the cycle and are executed at the time of expected division . After return to the permissive temperature, cells gradually recover from the temperature pulse as seen by a decrease in division delay . Preparation for the next division is unaffected by the temperature pulse . It occurs at the same time as in untreated controls . The results allow us to describe some features of the division initiating system. EMBO J, 1989 Mar, 8(3), 933 - 8 Identifying functional regions of rRNA by insertion mutagenesis and complete gene replacement in Tetrahymena thermophila; Sweeney R et al.; The free, linear macronuclear ribosomal RNA genes (rDNA) of Tetrahymena are derived from a unique copy of micronuclear rDNA during development . We have injected cloned copies of the micronuclear rDNA that have been altered in vitro into developing macronuclei and obtained transformants that express the paromomycin-resistant phenotype specified by the injected rDNA . In most cases, these transformants contain almost exclusively the injected rDNA which has been accurately processed into macronuclear rDNA . Mutants with a 119 bp insertion at three points in the transcribed spacers and at two points in the 26S rRNA coding region were tested . Cells containing these spacer mutant rDNAs are viable, although one of them grows slowly . This slow-growing line contains the insertion between the 5.8S and 26S rRNA coding regions and accumulates more rRNA processing intermediates than control lines . One of the 26S rRNA mutants failed to generate transformants, but the other did . These transformants grew normally, and produced 26S rRNA containing the inserted sequence . A longer insertion (2.3 kb) at the same four points either abolished transformation or generated transformants that retained at least some wild-type rDNA . This study reveals that some rRNA sequences can be altered without significantly affecting cell growth. Mikrobiol Zh, 1989 Mar-Apr, 51(2), 47 - 52 {Aminopeptidase from a thermophilic strain of Bacillus licheniformis}; Pavlova IN et al.; Aminopeptidase is isolated and purified from the culture liquid of the thermophilic strain of Bacillus licheniformis . The aminopeptidase predominantly splits off N-terminal leucin in short peptides and hydrolyzes leucinamide as well . The molecular weight of the enzyme is about 60 kDa . The enzyme is able to form aggregates . Optimum of aminopeptidase activity was demonstrated at pH 8.0-8.3 and temperature of 85 degrees C . The enzyme is inactivated by metal-binding reagents and reducing substances, and is activated by cobalt and PCMB ions . The EDTA-inactivated enzyme activity is reduced by cobalt and zinc ions, however the latter has no activating action . The enzyme under study is characterized by high thermostability: in the presence of the substrate at the temperature of 90 degrees C the reaction linearity is retained for not less than 2 h and without the substrate the half-life of the aminopeptidase at 90 degrees C is 145 min . Extracellular aminopeptidase of the thermophilic strain of B . licheniformis is a new enzyme differing from the aminopeptidases described by the present in high thermostability, induced, evidently, by the presence of one or several disulphide bonds in the enzyme molecule. J Bacteriol, 1989 Mar, 171(3), 1638 - 43 Coupled enzymatic production of sulfite, thiosulfate, and hydrogen sulfide from sulfur: purification and properties of a sulfur oxygenase reductase from the facultatively anaerobic archaebacterium Desulfurolobus ambivalens; Kletzin A; From aerobically grown cells of the extremely thermophilic, facultatively anaerobic chemolithoautotrophic archaebacterium Desulfurolobus ambivalens (DSM 3772), a soluble oxygenase reductase (SOR) was purified which was not detectable in anaerobically grown cells . In the presence of oxygen but not under a hydrogen atmosphere, the enzyme simultaneously produced sulfite, thiosulfate, and hydrogen sulfide from sulfur . Nonenzymatic control experiments showed that thiosulfate was produced mainly in a chemical reaction between sulfite and sulfur . The maximum specific activity of the purified SOR in sulfite production was 10.6 mumol/mg of protein at pH 7.4 and 85 degrees C . The ratio of sulfite to hydrogen sulfide production was 5:4 in the presence of zinc ions . The temperature range of enzyme activity was 50 to 108 degrees C, with a maximum at 85 degrees C . The molecular mass of the native SOR was 550 kilodaltons, determined by gel filtration . It consisted of identical subunits with an apparent molecular mass of 40 kilodaltons in sodium dodecyl sulfate-gel electrophoresis . The particle diameter in electron micrographs was 15 /+- 1.5 nm . The enzyme activity was inhibited by the thiol-binding reagents p-chloromercuribenzoic acid, N-ethyl maleimide, and 2-iodoacetic acid and by flavin adenine dinucleotide, Fe3+, and Fe2+ . It was not affected by CN-, N3-, or reduced glutathione. Development, 1989 Mar, 105(3), 457 - 71 Maintenance and regulation of cellular handedness in Tetrahymena; Nelsen EM et al.; The left-handed phenotype of Tetrahymena thermophila (LH) is a global mirror image of its right-handed counterpart (RH) . LH cells are 'wound' in the opposite direction from that of RH cells with respect to the placement of all structures that are asymmetrically disposed on the cell circumference . However, the local geometry of ciliary rows, including the asymmetrically placed microtubule bands and other accessory structures, is identical in RH and LH cells . Populations of LH cells grow more slowly than those of RH cells, probably because of nutritional problems due to faulty construction of the cell mouth . LH cells, like RH cells, conjugate in a homopolar configuration, while LH cells mate with RH cells in a heteropolar union which suffices to initiate the conjugal nuclear events but is insufficient to allow survival of progeny . Subclonal analyses indicate that reversion of the LH to the RH form is relatively rare . However, the frequency of reversion is greatly increased by conditions that promote the formation of doublets by fission arrest . An analysis of intermediate doublet forms in such cultures strongly suggests that reversion takes place through a specific pathway, with LH-LH doublets regulating to LH-RH forms that then may give rise to RH singlets . The origin and fate of the LH-RH intermediate forms can be explained by applying a modified polar coordinate model of positional information with the proviso that there is a preferred direction for the intercalation of new positional values. FEBS Lett, 1989 Feb 27, 244(2), 391 - 6 Low-molecular-mass proteins in cyanobacterial photosystem II: identification of psbH and psbK gene products by N-terminal sequencing; Koike H et al.; The O2-evolving photosystem II core complex was isolated from a thermophilic cyanobacterium, Synechococcus vulcanus Copeland . Analysis by SDS-polyacrylamide gel electrophoresis revealed that the complex contained at least seven low-molecular-mass proteins in addition to the well characterized CP47 apoprotein, CP43 apoprotein, 33 kDa extrinsic protein, D1 protein, D2 protein and large subunit of cytochrome b-559 . The separation of these low-molecular-mass proteins were very similar between cyanobacterial and higher plant PS II . N-terminal sequences of the 6.5 kDa and 3.9 kDa proteins of cyanobacterial core complex were determined after blotting to a polyvinylidene difluoride membrane . The sequence of the 6.5 kDa protein showed high homology with an internal sequence of plant psbH gene product, so-called 10 kDa phosphoprotein, but did not conserve the Thr residue which is specifically phosphorylated in plants . The sequence of the 3.9 kDa protein corresponded to the K protein of higher plants (mature form of psbK gene product) . These results indicate that the products of both psbH and psbK genes are present in cyanobacterial PS II as well as being associated with the O2-evolving core complex. Biochim Biophys Acta, 1989 Feb 24, 990(2), 133 - 7 A highly stable NADP-dependent isocitrate dehydrogenase from Thermus thermophilus HB8: purification and general properties; Eguchi H et al.; NADP-dependent isocitrate dehydrogenase (EC 1.1.1.42) was purified to electrophoretic homogeneity from an extremely thermophilic bacterium, Thermus thermophilus HB8, and shown to be a dimeric protein of molecular weight 115,000, with a pI of 5.5 . The amino acid composition of the present enzyme was similar to that reported for other bacterial counterparts, except for a high Arg/Lys ratio and a low Cys content . Divalent cations, such as Mn2+ and Mg2+, were essential for activity . The optimal pH was 7.8 at 55 degrees C . The Km values for NADP and D-isocitrate were 6.3 and 8.8 microM, respectively, with a Vmax of 77.6 mumol/min per mg at 55 degrees C . NAD was able to replace NADP with low efficiency . Backward reaction at 40 degrees C indicated that the Km value for 2-oxoglutarate was 63 microM with a Vmax of 4% that of the forward reaction at that temperature . The enzyme was highly stable against high temperature and denaturing reagents. Biochim Biophys Acta, 1989 Feb 23, 994(3), 270 - 9 Isolation and characterisation of the glycerol dehydrogenase from Bacillus stearothermophilus; Spencer P et al.; A protocol for the rapid purification of the glycerol dehydrogenase (glycerol: NAD+ 2-oxidoreductase, EC 1.1.1.6) from the thermophile Bacillus stearothermophilus has been developed using a combination of chromatographic techniques including affinity chromatography on a Sepharose-immobilised triazine dye (Procion red, HE3B, ICI) . Substrate specificity has been examined and Km values determined . The protein has been shown to have an oligomeric Mr of approx . 180,000 and consists of four identical subunits of Mr 42,000 . Exposure to chelating agents (e.g., EDTA) leads to total loss of activity; the EDTA-inactivated enzyme can be reactivated by Zn2+ and requires 1 mol equivalent of zinc per subunit for full catalytic activity . Other divalent cations such as Cd2+ and Co2+ will reactivate the apo-enzyme but yields an enzyme of lower specific activity . The enzyme binds 1 equivalent of NADH per subunit and during catalysis transfers the 4-pro-R hydride from the nicotinamide ring of the reduced-coenzyme to the substrate . Glycerol increases the dissociation constant for the interaction between NADH and Zn-metallo-glycerol dehydrogenase (ZnGDH) but has no effect on the equilibrium between NADH and metal-depleted enzyme. Nature, 1989 Feb 16, 337(6208), 655 - 9 Characterization of the yeast HSP60 gene coding for a mitochondrial assembly factor; Reading DS et al.; The hsp60 protein isolated from the protozoan Tetrahymena thermophila is induced in response to heat stress and is a member of an immunologically conserved family represented in Escherichia coli and in mitochondria of plants and animals . We report here the cloning and characterization of a nuclear gene, HSP60, which codes for the hsp60 homologue from the yeast Saccharomyces cerevisiae . Nucleotide sequence analysis revealed that yeast hsp60 is related to the groEL protein of E . coli and the RUBISCO-binding protein (RBP) of chloroplasts . HSP60 was found to be the genetic locus of the conditional-lethal mutation described by Cheng et al., which at non-permissive temperature is defective in the assembly of several different multisubunit complexes in mitochondria . These data are consistent with the hypothesis that the groEL-related proteins serve an evolutionarily conserved function as accessory factors facilitating the folding and/or association of individual subunits of multimeric protein complexes. J Biol Chem, 1989 Feb 15, 264(5), 2445 - 9 Thermostable D-amino acid aminotransferase from a thermophilic Bacillus species . Purification, characterization, and active site sequence determination; Tanizawa K et al.; D-Amino acid aminotransferase was found in several thermophilic Bacillus species and purified to homogeneity from the best producer, Bacillus sp . YM-1, which was newly isolated from a sauna dust . The enzyme has a molecular weight of about 62,000 and consists of two subunits identical in molecular weight (30,000) . It catalyzes transamination between various D-amino acids and alpha-keto acids, although the substrate specificity is narrower than the enzyme from the mesophile, Bacillus sphaericus (Yonaha, K., Misono, H., Yamamoto, T., and Soda, K . (1975) J . Biol . Chem . 250, 6983-6989) . The Bacillus sp . YM-1 enzyme is most active at 60 degrees C and stable at high temperatures . Automated Edman degradation provided the N-terminal sequence of the first 20 amino acids, and carboxypeptidase Y digestion provided the C-terminal sequence of the last 3 amino acids . The amino acid sequence in the vicinity of the lysyl residue, Lys(Pxy), that binds pyridoxal 5'-phosphate was determined as Cys-Asp-Ile-Lys(Pxy)-Ser-Leu-Asn-Leu-Leu-Gly-Ala-Val-Leu-Ala-Lys- from the pyridoxyl peptide obtained by digestion with trypsin . The active site sequence is markedly different from those of L-amino acid aminotransferases and other pyridoxal 5'-phosphate-dependent enzymes. J Biol Chem, 1989 Feb 15, 264(5), 2450 - 4 The primary structure of thermostable D-amino acid aminotransferase from a thermophilic Bacillus species and its correlation with L-amino acid aminotransferases; Tanizawa K et al.; The gene for thermostable D-amino acid aminotransferase from a thermophile, Bacillus species YM-1 was cloned and expressed efficiently in Escherichia coli . The entire covalent structure of the enzyme was determined from the nucleotide sequence of the cloned gene and mostly confirmed by amino acid sequences of tryptic peptides from the gene product . The polypeptide is composed of 282 amino acid residues with a calculated molecular weight of 32,226 . Comparison of the primary structure with those of various proteins registered in a protein data bank revealed a significant sequence homology between D-amino acid aminotransferase and the L-branched chain amino acid aminotransferase of E . coli (Kuramitsu, S., Ogawa, T., Ogawa, H., and Kagamiyama, H . (1985) J . Biochem . (Tokyo) 97, 993-999); the active site lysyl residue is located in an equivalent position in both enzyme sequences of similar size . Despite the difference in subunit composition and no immunochemical cross-reactivity, the sequences of the two enzymes show similar hydropathy profiles, and spectrophotometric properties of the enzyme-bound cofactor are also similar . The sequence homology suggests that the structural genes for D-amino acid and L-branched chain amino acid aminotransferases evolved from a common ancestral gene. J Biol Chem, 1989 Feb 15, 264(5), 2405 - 8 An infrared study of the binding and photodissociation of carbon monoxide in cytochrome ba3 from Thermus thermophilus; Einarsdottir O et al.; The C-O stretching frequencies of fully reduced carbonmonoxy cytochrome ba3, a newly discovered terminal oxidase of the bacterium Thermus thermophilus (Zimmermann, B.H., Nitsche, C.I., Fee, J.A., Rusnak, F., and Munck, E . (1988) Proc . Natl . Acad . Sci . U.S . A . 85, 5779-5783), are studied by Fourier transform infrared spectroscopy . Multiple C-O frequencies are observed in the Fourier transform infrared spectra, indicating the presence of discrete interconverting conformers of the enzyme . Upon photolysis, the CO is shown to migrate exclusively to CuB+ . Above 200 K, the CO returns to the heme a3 by a thermal process which follows simple first-order kinetics . The rate of the reaction was studied from 205 to 230 K and at 300 K, yielding the activation parameters delta H = 14.9 kcal/mol and delta S = -5 cal/mol/K . These are compared with previously determined activation parameters for CO recombination in mitochondrial cytochrome aa3 preparations (Fiamingo, F.G., Altschuld, R.A., Moh, P.P., and Alben, J.O . (1982) J . Biol . Chem . 257, 1639-1650) . We report the novel finding that CO remains bound to CuB+ at room temperature during continuous photolysis of cytochrome ba3, and we conjecture on the possible interference of copper-bound CO in "flow-flash" and "triple-trap" studies of cytochrome c oxidases. Nucleic Acids Res, 1989 Feb 11, 17(3), 845 - 51 Structural and functional exchangeability of 5 S RNA species from the eubacterium E.coli and the thermoacidophilic archaebacterium Sulfolobus solfataricus; Teixido J et al.; The role of 5 S RNA within the large ribosomal subunit of the extremely thermophilic archaebacterium Sulfolobus solfataricus has been analysed by means of in vitro reconstitution procedures . It is shown that Sulfolobus 50 S subunits reconstituted in the absence of 5 S RNA are inactive in protein synthesis and lack 2-3 ribosomal proteins . Furthermore, it has been determined that in the course of the in vitro assembly process Sulfolobus 5 S RNA can be replaced by the correspondent RNA species of E.coli; Sulfolobus reconstituted particles containing the eubacterial 5 S molecule are stable and active in polypeptide synthesis at high temperatures. Mol Cell Biol, 1989 Feb, 9(2), 828 - 30 Methylation of replicating and nonreplicating DNA in the ciliate Tetrahymena thermophila; Harrison GS et al.; Methylation of adenine in replicating and nonreplicating DNA of the ciliate Tetrahymena thermophila was examined . In growing cells, 87% of the methylation occurred on the newly replicated daughter strand, but methylation was also detectable on the parental strand . Methylation of nonreplicating DNA from starved cells was demonstrated. Mol Cell Biol, 1989 Feb, 9(2), 452 - 60 The replication advantage of a free linear rRNA gene is restored by somatic recombination in Tetrahymena thermophila; Yaeger PC et al.; The autonomously replicating rRNA genes (rDNA) in the somatic nucleus of Tetrahymena thermophila are maintained at a copy number of approximately 10(4) per nucleus . A mutant in which the replication properties of this molecule were altered was isolated and characterized . This mutation of inbred strain C3, named rmm4, was shown to have the same effect on rDNA replication and to be associated with the same 1-base-pair (bp) deletion as the previously reported, independently derived rmm1 mutation (D . L . Larson, E . H . Blackburn, P . C . Yaeger, and E . Orias, Cell 47:229-240, 1986) . The rDNA of inbred strain B, which is at a replicational disadvantage compared with wild-type C3 rDNA, has a 42-bp deletion . This deletion is separated by 25 bp from the 1-bp deletion of rmm4 or rmm1 . Southern blot analysis and DNA sequencing revealed that during prolonged vegetative divisions of C3-rmm4/B-rmm heterozygotes, somatic recombination produced rDNAs lacking both the rmm4-associated deletion and the 42-bp deletion . In somatic nuclei in which this rare recombinational event had occurred, all 10(4) copies of nonrecombinant rDNA were eventually replaced by the recombinant rDNA . The results prove that each of the two deletions is the genetic determinant of the observed replication disadvantage . We propose that the analysis of somatically recombinant rDNAs can be used as a general method in locating other mutations which affect rDNA propagation in T . thermophilia. Curr Genet, 1989 Feb, 15(2), 99 - 106 Isolation and complete sequence of the yeast isoleucyl-tRNA synthetase gene (ILS1); Martindale DW et al.; The isoleucyl-tRNA synthetase gene (ILS1) from the yeast Saccharomyces cerevisiae was cloned and sequenced . This gene was initially cloned because it cross-hybridizated to what is now presumed to be the isoleucyl-tRNA synthetase gene (cupC) from the protozoan Tetrahymena thermophila . The ILS1 gene was determined to be 1,072 amino acids in length . A comparison with a recently published sequence of ILS1 from another laboratory (Englisch et al . 1987) was made and differences noted . Two promoter elements were detected, one for general amino acid control and one for constitutive transcription . A heat shock protein (hsp70) gene (probably SSA3) was found 237 bp upstream from the ILS1 translation start site . The ILS1 amino acid sequence was compared to isoleucyl-tRNA synthetases from other organisms, as well as to valyl-, leucyl- and methionyl-tRNA synthetases . Regions of conservation between these enzymes were found. J Bacteriol, 1989 Feb, 171(2), 1219 - 22 Cloning and expression in Escherichia coli of an extremely thermostable oligo-1,6-glucosidase gene from Bacillus thermoglucosidasius; Watanabe K et al.; The gene for an extremely thermostable oligo-1,6-glucosidase (dextrin-6-alpha-D-glucanohydrolase; EC 3.2.1.10) of obligately thermophilic Bacillus thermoglucosidasius KP1006 was cloned within a 4.2-kilobase HindIII-PvuII fragment of DNA by using the plasmid pUC19 as a vector and Escherichia coli C600 as a host . The gene was transcribed, presumably from its own promoter, in E . coli . E . coli with the hybrid plasmid accumulated oligo-1,6-glucosidase mainly in the cytoplasm . The level of enzyme production was comparable to that observed for B . thermoglucosidasius . The enzyme coincided absolutely with the B . thermoglucosidasius enzyme in its molecular weight (60,000), in its electrophoretic behavior on denaturing and nondenaturing polyacrylamide gels, in the temperature dependency of its stability and activity, and in its antigenic determinants. J Gen Microbiol, 1989 Feb, 135 ( Pt 2), 361 - 73 Development of monoclonal-antibody-ELISA, -DOT-BLOT and -DIP-STICK immunoassays for Humicola lanuginosa in rice; Dewey FM et al.; Monoclonal antibodies (MAbs) were raised against Humicola lanuginosa, a thermophilic, saprophytic fungus that is commonly isolated from freshly harvested rice grains in Indonesia . Mice were immunized by direct injection into the peritoneum, without prior concentration, of fresh cell-free surface washings from a solid agar slant culture . Hybridoma supernatants were screened by ELISA using wells coated with a dilution of the immunogen . From one fusion 403 hybridoma clones were obtained yielding 52 cell lines secreting antibodies positive for H . lanuginosa . Twelve cell lines were re-cloned, grown in bulk and tested against other storage fungi . Most of the MAbs raised were IgM antibodies that cross-reacted with several of the storage fungi and/or uninfected rice grains . However, the IgM antibody EC6 did not recognize antigens from rice grains and cross-reacted strongly with only one other test fungus, Penicillium variabile, and partially with two others . This MAb was used to develop a highly sensitive DIP-STICK immunoassay to detect the fungus in infected grains . These assays are simple to perform, require no equipment and are suitable for field use by untrained workers. J Dairy Res, 1989 Feb, 56(1), 107 - 16 Uptake of glutamic acid by Streptococcus salivarius subsp . thermophilus CNRZ 302; Bracquart P et al.; Glutamic acid uptake in Streptococcus salivarius subsp . thermophilus was energy dependent, the source of energy and adaptation to sugar being important to efficiency of uptake . The disaccharides, lactose and sucrose, stimulated uptake, but cells grown in glucose were more active . Optimum temperature was approximately 40 degrees C and pH approximately 7.0 . NaCl was strongly inhibitory to the uptake of glutamic acid although not to that of isoleucine . High specificity existed because only L-aspartic acid was inhibitory. Poult Sci, 1989 Feb, 68(2), 311 - 4 Population of Salmonella typhimurium in the cecum of gnotobiotic chickens; Fukata T et al.; To test the interaction of various species of bacteria with Salmonella typhimurium, levels of S . typhimurium were measured in the cecum of gnotobiotic chickens inoculated with various test bacteria and subsequently with S . typhimurium . The population of S . typhimurium was suppressed in the cecum of chickens previously inoculated with Escherichia coli . Populations of Bacteroides vulgatus, Clostridium perfringens, Bifidobacterium thermophilum, or Lactobacillus acidophilus were transiently, though not significantly, suppressed by S . typhimurium . Inoculation of chickens with C . perfringens caused a terminal increase in the population of S . typhimurium . Thus the effect of cecal contents on reducing the levels of S . typhimurium in the cecum may be due to the presence of E . coli in the cecal contents. J Med Virol, 1989 Feb, 27(2), 105 - 11 Polymerase chain reaction for fast, nonradioactive detection of high- and low-risk papillomavirus types in routine cervical specimens and in biopsies; Dallas PB et al.; A polymerase chain reaction (PCR) procedure capable of amplifying specific DNA sequences by up to a millionfold was developed for detection of infection by human papillomaviruses (HPVs) of low (HPV6, HPV11) or high (HPV16, HPV18, HPV33) oncogenic potential . For high-risk HPVs the region chosen was within the E6 open reading frame, which can become integrated into genomic DNA . A region corresponding to this was chosen for low-risk HPVs . After repeated cycles of specific oligonucleotide-primed extension of viral DNA with Klenow or thermophilic DNA polymerase, the type of HPV present was then determined on the basis of the size of the ethidium-bromide-stained band visible after polyacrylamide gel electrophoresis: for HPV6 or 11 the band was approximately 120 bp, for HPV 16 or 33 it was approximately 200 bp, and for HPV18 it was approximately 100 bp . Specific hybridization to the relevant band was seen using radioactive or nonradioactive (alkaline-phosphatase-linked) target oligonucleotide probes . Using the PCR method, we have determined, within as little as a few hours, the infection status of a variety of clinical specimens, including cervical scrapes and lavages, anal scrapes, and anogenital biopsies . The PCR steps can be automated, adding to the potential of PCR for widespread use in the detection of HPV, which is becoming increasingly popular in cervical screening. J Dairy Sci, 1989 Feb, 72(2), 351 - 9 A method for determining beta-galactosidase activity of yogurt cultures in skim milk; Lin WJ et al.; A method was developed for determining the specific activity of bacterial beta-galactosidase (EC 3.2.1.23) during growth of Streptococcus thermophilus and Lactobacillus bulgaricus in skim milk . Individual and mixed strain cultures of S . thermophilus (St 3642, St14485) and L . bulgaricus (Lb11842, Lb880) were examined for growth (OD at 600 nm and viable cell counts), acid production, and beta-galactosidase activity (expressed as a function of recoverable TCA-precipitable cellular protein) . Cultures were inoculated into 10% skim milk (2% inoculum) and incubated at 40 degrees C for 12 h . Aliquots were removed at 2-h intervals and diluted with ice cold EDTA, pH 12 . The EDTA chelates calcium and solubilizes milk protein, allowing separation of the bacteria by centrifugation . Cells were then washed twice with 20 mM phosphate buffer and disrupted by sonication . Cell debris and intact cells were removed by centrifugation and the cell-free extract evaluated for beta-galactosidase activity using o-nitrophenyl-beta-D-galactopyranoside as substrate . Specific activities ranged from 0 to 6 units/mg protein . This simple and reproducible method is applicable for enzyme assays and measurement of cellular components where contamination by milk proteins is a potential problem. J Dairy Res, 1989 Feb, 56(1), 117 - 27 Purification and thermostability of beta-galactosidase (lactase) from an autolytic strain of Streptococcus salivarius subsp . thermophilus; Chang BS et al.; beta-Galactosidase from an autolytic strain of Streptococcus salivarius subsp . thermophilus was purified 109-fold to near homogeneity . The yield of purified enzyme was 41% and the specific activity was 592 o-nitrophenyl beta-D-galactopyranoside U/mg at 37 degrees C . Two isozymes were present, but only one subunit was detected, having a mol . wt of 116,000 . Enzyme stability was 37-83 times greater in milk than in buffer in the range 60-65 degrees C . At 60 degrees C the half-life in milk was 146 min . Denaturation in buffer was first-order, but in milk the overall reaction order with respect to enzyme concentration was approximately 0.5 . The activation energy for denaturation was 453 kJ/mol in milk and 372 kJ/mol in buffer . In milk the activation energy for lactose hydrolysis was 35.1 kJ/mol. Eur J Biochem, 1989 Feb 1, 179(2), 405 - 13 Nucleotide sequence of the glyceraldehyde-3-phosphate dehydrogenase gene from the mesophilic methanogenic archaebacteria Methanobacterium bryantii and Methanobacterium formicicum . Comparison with the respective gene structure of the closely related extreme thermophile Methanothermus fervidus; Fabry S et al.; The genes for glyceraldehyde-3-phosphate dehydrogenase (gap genes) from the mesophilic methanogenic archaebacteria Methanobacterium formicicum and Methanobacterium bryantii were cloned and sequenced . The deduced amino acid sequences show 95% identity to each other and about 70% identity to the glyceraldehyde-3-phosphate dehydrogenase from the thermophilic methanogenic archaebacterium Methanothermus fervidus . Although the sequence similarity between the archaebacterial glyceraldehyde-3-phosphate dehydrogenase and the homologous enzyme of eubacteria and eukaryotes is low, an equivalent secondary-structural arrangement can be deduced from the profiles of the physical parameters hydropathy, chain flexibility and amphipathy . In order to find possible thermophile-specific structural features of the enzyme from M . fervidus, a comparative primary-sequence analysis was performed . Amino acid exchanges leading, to a stabilization of the main-chain conformation, could be found throughout the sequence of the thermophile enzyme . Striking features of the thermophile sequence are the preference for isoleucine, especially in beta-sheets, and a low arginine/lysine ratio of 0.54. J Bacteriol, 1989 Feb, 171(2), 625 - 35 Expression and nucleotide sequence of the Lactobacillus bulgaricus beta-galactosidase gene cloned in Escherichia coli; Schmidt BF et al.; The Lactobacillus bulgaricus beta-galactosidase gene was cloned on a ca . 7-kilobase-pair HindIII fragment in the vector pKK223-3 and expressed in Escherichia coli by using its own promoter . The nucleotide sequence of the gene and approximately 400 bases of 3'- and 5'-flanking sequences was determined . The amino acid sequence of the beta-galactosidase, deduced from the nucleotide sequence of the gene, yielded a monomeric molecular mass of ca . 114 kilodaltons, slightly smaller than the E . coli lacZ and Klebsiella pneumoniae lacZ enzymes but larger than the E . coli evolved (ebgA) beta-galactosidase . The cloned beta-galactosidase was found to be indistinguishable from the native enzyme by several criteria . From amino acid sequence alignments, the L . bulgaricus beta-galactosidase has a 30 to 34% similarity to the E . coli lacZ, E . coli ebgA, and K . pneumoniae lacZ enzymes . There are seven regions of high similarity common to all four of these beta-galactosidases . Also, the putative active-site residues (Glu-461 and Tyr-503 in the E . coli lacZ beta-galactosidase) are conserved in the L . bulgaricus enzyme as well as in the other two beta-galactosidases mentioned above . The conservation of active-site amino acids and the large regions of similarity suggest that all four of these beta-galactosidases evolved from a common ancestral gene . However, these enzymes are quite different from the thermophilic beta-galactosidase encoded by the Bacillus stearothermophilus bgaB gene. Chem Pharm Bull (Tokyo), 1989 Feb, 37(2), 284 - 91 Studies towards the synthesis of the fluorescent bases of phenylalanine transfer ribonucleic acids: synthesis of 7-methylwye isolated from extremely thermophilic archaebacteria; Itaya T et al.; Acid- or base-catalyzed acylation of 1-benzylwye (7) provided the 7-substituted derivatives 9, 10, and 11 in poor yields . Although the reactions of lithiated 7 with electrophiles gave the 2-substituted derivatives 14, 15, 17, 20, 21, and 22, lithiation of 1-benzyl-7-bromo-2-chlorowye (23) followed by treatment with Me2CHCH2CHO (13) successfully introduced a side chain at the 7-position to afford 1-benzyl-2-chloro-7-(1-hydroxy-3-methylbutyl)wye (24) . Cyclization of 1-benzyl-3-methylguanine (5) with 3-bromo-2-butanone followed by catalytic hydrogenolysis afforded 7-methylwye (2b), the hypermodified base isolated from archaebacterial transfer ribonucleic acids . A more efficient route for the synthesis of 2b has been developed via a series of reactions: the Vilsmeier-Haack reaction of 7, reduction with NaBH4, and catalytic hydrogenolysis over Pd-C. Nucleic Acids Res, 1989 Jan 25, 17(2), 675 - 89 Compensatory mutations demonstrate that P8 and P6 are RNA secondary structure elements important for processing of a group I intron; Williamson CL et al.; Compensatory mutations have been constructed which demonstrate that P8 and P6, two of nine proposed base-pairing interactions characteristic of group I introns, exist within the folded structure of the Tetrahymena thermophila rRNA intervening sequence, and that these secondary structure elements are important for splicing in E . coli and self-splicing in vitro . Two-base mutations in the 5' and 3' segments of P8 are predicted to disrupt P8 and a strong splicing-defective phenotype is observed in each case . A compensatory four-base mutation in P8 is predicted to restore pairing, and results in the restoration of splicing activity to nearly wild type levels . Thus, we conclude that P8 exists and is essential for splicing . In contrast to the strong phenotypes generally exhibited by mutations which disrupt RNA secondary structure, a two-base mutation in L8, the loop between P8{5'} and P8{3'}, results in only a slight decrease in splicing activity . We also tested P6, a pairing which is proposed to consist of only two base-pairs in this intron . A two-base mutation in P6{3'} reduces splicing activity to a greater extent than does a two-base mutation in P6{5'} . Comparison of the activities of these mutants and a compensatory P6 four-base mutant support the existence of P6, and suggest that the P6 pairing may be particularly important in the exon ligation step of splicing. Biochemistry, 1989 Jan 24, 28(2), 505 - 9 Site-directed mutagenesis of the cysteinyl residues and the active-site serine residue of bacterial D-amino acid transaminase; Merola M et al.; Each of the three cysteinyl residues per subunit in D-amino acid transaminase from a thermophilic species of Bacillus has been changed to a glycine residue (C142G, C164G, and C212G) by site-directed mutagenesis . The mutant enzymes were detected by Western blots and a stain for activity . After purification to homogeneity, each mutant protein had the same activity as the wild-type enzyme . Thus, none of the Cys residues are essential for catalysis . Each protein when denatured showed the expected titer of two SH groups per subunit . In the native state, each of the three mutant proteins exhibited nearly the same slow rate of titration of SH groups as the wild-type protein with about one SH group titratable over a period of 4 h . Conversion of Ser-146, adjacent to Lys-145 to which the coenzyme pyridoxal phosphate is bound, to an alanine residue (S146A) does not alter the catalytic activity but has a significant effect on the SH titration behavior . Thus, three to four of the six SH groups of S146A are titratable by DTNB . The rapid SH titration of S146A is prevented by the presence of D-alanine . This finding suggests that the change of Ser-146 to Ala at the active site promotes the exposure and rapid titration of a Cys residue in that region . The rapid SH titration of S146A by DTNB is accompanied by a loss of enzyme activity . Two of the mutant enzymes, C142G and S146A, lose activity at 4 degrees C and also upon freezing and thawing . The mutant enzymes C164G and C212G show the same degree of thermostability as the wild-type enzyme. Nucleic Acids Res, 1989 Jan 11, 17(1), 355 - 71 Effects of substrate structure on the kinetics of circle opening reactions of the self-splicing intervening sequence from Tetrahymena thermophila: evidence for substrate and Mg2+ binding interactions; Sugimoto N et al.; The self-splicing intervening sequence from the precursor rRNA of Tetrahymena thermophila cyclizes to form a covalently closed circle . This circle can be reopened by reaction with oligonucleotides or water . The kinetics of circle opening as a function of substrate and Mg2+ concentrations have been measured for dCrU, rCdU, dCdT, and H2O addition . Comparisons with previous results for rCrU suggest: (1) the 2' OH of the 5' sugar of a dinucleoside phosphate is involved in substrate binding, and (2) the 2' OH of the 3' sugar of a dimer substrate is involved in Mg2+ binding . Evidently, the binding site for a required Mg2+ ion is dependent on both the ribozyme and the dimer substrate . The apparent activation energy and entropy for circle opening by hydrolysis are 31 kcal/mol and 50 eu, respectively . The large, positive activation entropy suggests a partial unfolding of the ribozyme is required for reaction. Dev Biol, 1989 Jan, 131(1), 261 - 8 Cloning of Tetrahymena genomic sequences whose message abundance is increased during conjugation; Rogers MB et al.; A molecular and biochemical inquiry into protein regulation during Tetrahymena thermophila conjugation was carried out in two ways: a two-dimensional gel analysis of newly translated proteins and the molecular cloning of genes whose message abundance is increased . The two-dimensional gel analysis indicated that the synthesis of 32 predominantly basic proteins was stimulated in conjugating cells . The induction of these proteins could not be correlated with length of starvation or with mating type . The transcription pattern and molecular organization of three clones of T . thermophila genomic DNA, selected on the basis of differential hybridization to conjugating or control cell RNA, were investigated . Two of the clones, which were homologous to transcripts detected in conjugating cells, showed no rearrangements between micro- and macronuclear DNA . A third clone was divided into three segments . One segment was homologous to sequences limited to the micronucleus . A second segment hybridized to a large number of restriction fragments of micronuclear DNA digested with HindIII but to only two fragments of macronuclear DNA . A third segment, which was complementary to one transcript in conjugating cells and to two different transcripts in control cells, hybridized to two fragments in micronuclear DNA and one fragment in macronuclear DNA. Intervirology, 1989, 30(4), 237 - 40 Distribution of homologies among the genomes of several actinophages of Faenia and Saccharopolyspora as determined by DNA hybridization; Schneider J et al.; The DNA of phi FR114, a temperate phage of the thermophilic actinomycete Faenia rectivirgula, was hybridized with the genomes of 11 different phages of Faenia and Saccharopolyspora . This revealed several regions (modules) within the phi FR114 DNA which were distributed independently among the genomes of the other phages . The genome of the lytic phage 121, originally described for Sap . erythraea, exhibited a similar modular organization . So far no functions of the possible modules are known. Antonie Van Leeuwenhoek, 1989, 55(2), 153 - 63 Isolation and characterization of xylose- and xylan-utilizing anaerobic bacteria; Murty MV et al.; By enrichment with xylose, nine mesophilic strains of anaerobic bacteria were obtained from various sources . Two isolates appear to belong to the genus Eubacterium . Six other strains belong to the genus Clostridium . Three of the isolated strains utilized larch wood xylan . The percentage of utilization of xylose and xylan and the yield of fermentation end products--viz . acetic acid and butyric acid--are equivalent to that of Clostridium acetobutylicum (ATCC 824) and reported thermophilic strains. Can J Microbiol, 1989 Jan, 35(1), 109 - 18 Origin of the eukaryotic nucleus: eukaryotes and eocytes are genotypically related; Lake JA; The origin of the eukaryotic nucleus is difficult to reconstruct . While eukaryotic organelles (chloroplast, mitochondrion) are eubacterial endosymbionts, the source of nuclear genes has been obscured by multiple nucleotide substitutions . Using evolutionary parsimony, a newly developed rate-invariant treeing algorithm, the eukaryotic rRNA genes are shown to have evolved from the eocytes, a group of extremely thermophilic, sulfur-metabolizing, anucleate cells . The deepest bifurcation yet found separates the reconstructed tree into two taxonomic divisions . These are a proto-eukaryotic group (karyotes) and an essentially bacterial one (parkaryotes) . Within the precision of the rooting procedure, the tree is not consistent with either the prokaryotic--eukaryotic or the archaebacterial--eubacterial--eukaryotic groupings . It implies that the last common ancestor of extant life, and the early ancestors of eukaryotes, very likely lacked nuclei, metabolized sulfur, and lived at near boiling temperatures. Anal Chem, 1989 Jan 1, 61(1), 25 - 9 Thermostable reduced nicotinamide adenine dinucleotide oxidase: application to amperometric enzyme assay; McNeil CJ et al.; The use in amperometric enzyme assays of a highly stable, pH insensitive flavoenzyme, reduced nicotinamide adenine dinucleotide oxidase (NADH oxidase), from the thermophilic organism Thermus aquaticus is described . The enzyme catalyses the oxidation of reduced nicotinamide adenine dinucleotide with concomitant two-electron reduction of dioxygen to hydrogen peroxide . In addition the enzyme used a substituted ferrocene as an alternative mediator of electron transfer . Hydrogen peroxide was detected at +650 mV vs Ag/AgCl at a platinum electrode . The current produced by oxidation of hydrogen peroxide was directly proportional to NADH concentration . The enzyme was used in solution to reoxidize enzymatically generated NADH and served as a basis for amperometric enzyme amplification systems for immunoassay as well as for the detection of substrate concentration for oxidoreductase enzymes . In the presence of alcohol dehydrogenase a rapid production of current occurred upon addition of ethanol over a clinically significant range . Thermus aquaticus NADH oxidase appears to be ideally suited for future exploitation in amperometric sensors for oxidoreductase substrates, offering a number of advantages over previously reported methods. J Protozool, 1989 Jan-Feb, 36(1), 92 - 5 Glutathione in Tetrahymena thermophila; Wagner B; In Tetrahymena, glutathione is synthesized from the same precursors as it is in higher animals and is present in similar intracellular concentrations . The intracellular thiol-disulfide ratio is also identical to that of mammalian tissues, due to the activity of glutathione reductase . The intracellular GSH-level was found to be dependent on the sulfur-containing amino acids in the chemically defined medium. Z Erkr Atmungsorgane, 1989, 173(2), 151 - 60 {Allergic alveolitis in agricultural workers, caused by thermophilic bacteria or fungi}; Barzo P et al.; Between 1976-1986 fifty-seven patients with farmer's lung have been diagnosed in Hungary on the basis of data obtained from public institutes for tuberculosis . That are 0.08% of the 744,300 manual workers employed in agriculture and forestry . In the counties of Borsod and Szolnok the rate was 0.5% (referred to 62,900 and 36,000 individuals, respectively) . Regional accumulation of the different provoking agent's and variation of disease prevalence seem to be in correlation with geographical, climatic, meteorological, economical and occupational factors, showing a declining tendency in recent years . Antibodies of the Thermoactinomyces vulgaris antigen could be detected most frequently . In the biopsy material of 17 patients obtained by Klassen biopsy were fibrosing and not fibrosing desquamative alveolitis, granulomas similar to sarcoidosis, bronchiolitis with peribronchial fibrosis observed . Mostly focal, rarely subpleural deficiencies were detected by lung-scintigraphy, their dimension in acute cases being greater than the extent of radiological lesions . On the basis of 5 Coombs positive cases authors consider conceivable that sometimes cytotoxic allergic reaction type II participates in the pathogenesis of farmer's lung. Orig Life Evol Biosph, 1989, 19(2), 95 - 108 Sulfur, ultraviolet radiation, and the early evolution of life; Kasting JF et al.; The present biosphere is shielded from harmful solar near ultraviolet (UV) radiation by atmospheric ozone . We suggest here that elemental sulfur vapor could have played a similar role in an anoxic, ozone-free, primitive atmosphere . Sulfur vapor would have been produced photochemically from volcanogenic SO2 and H2S . It is composed of ring molecules, primarily S8, that absorb strongly throughout the near UV, yet are expected to be relatively stable against photolysis and chemical attack . It is also insoluble in water and would thus have been immune to rainout or surface deposition over the oceans . The concentration of S8 in the primitive atmosphere would have been limited by its saturation vapor pressure, which is a strong function of temperature . Hence, it would have depended on the magnitude of the atmospheric greenhouse effect . Surface temperatures of 45 degrees C or higher, corresponding to carbon dioxide partial pressures exceeding 2 bars, are required to sustain an effective UV screen . Two additional requirements are that the ocean was saturated with sulfite and bisulfite, and that linear S8 chains must tend to reform rings faster than they are destroyed by photolysis . A warm, sulfur-rich, primitive atmosphere is consistent with inferences drawn from molecular phylogeny, which suggest that some of the earliest organisms were thermophilic bacteria that metabolized elemental sulfur. Cell Mol Biol, 1989, 35(3), 293 - 6 Studies into hormonal imprinting in a Tetrahymena thermophila mutant incapable of lysosomal enzyme secretion; Kovacs P et al.; Reexposure to insulin after primary interaction (hormonal imprinting) was followed by a binding increase in T . pyriformis and by a binding decrease in T . thermophila . The sec . mutant, MS-1 strain of T . thermophila, which is unable of lysosomal enzyme secretion, also showed a binding increase on a second exposure to insulin, from which it follows that alteration of the enzyme secretion, or other factors associated with mutation, accounted for reversion of the trend of imprinting . Thyrotropic hormone (TSH) also gave rise to a negative imprinting in T . thermophila, but did not alter the binding relations of the MS-1 mutant strain. Genetics, 1989 Jan, 121(1), 37 - 45 Arrest of micronuclear DNA replication during genomic exclusion in Tetrahymena produces haploid strains; Kaczanowski A et al.; Diploid cells of Tetrahymena thermophila were crossed to strain A*V, whose micronucleus is defective, to induce the unilateral transfer of gametic nuclei from the diploid cells to the A*V cells (round I of genomic exclusion) . These haploid nuclei presumably undergo one endomitotic cycle and then become diploid with a G1 (2C) DNA content . However, further DNA replication from 2C to 4C was transiently arrested until the pairs separated . When endomitosis was blocked by treatment with cycloheximide during 6-8 hours of conjugation, the exconjugants of round I of genomic exclusion remained haploid . Competence for diploidization is apparently limited to some period of time after nuclear transfer . Blocking of diploidization during round I of genomic exclusion can be used as an efficient way to induce haploid strains in Tetrahymena. J Bacteriol, 1989 Jan, 171(1), 244 - 53 Lactose transport system of Streptococcus thermophilus: a hybrid protein with homology to the melibiose carrier and enzyme III of phosphoenolpyruvate-dependent phosphotransferase systems; Poolman B et al.; The gene responsible for the transport of lactose into Streptococcus thermophilus (lacS) was cloned in Escherichia coli as a 4.2-kilobase fragment from an EcoRI library of chromosomal DNA by using the vector pKK223-3 . From deletion analysis, the gene for lactose transport mapped to two HindIII fragments with a total size of 2.8 kilobases . The gene was transcribed in E . coli from its own promoter . Functional expression of lactose transport activity was shown by assaying for the uptake and exchange of lactose both in intact cells and in membrane vesicles . The nucleotide sequence of lacS and 200 to 300 bases of 3' and 5' flanking regions were determined . The gene was 1,902 base pairs long, encoding a 69,454-dalton protein with an NH2-terminal hydrophobic region and a COOH-terminal hydrophilic region . The NH2-terminal end was homologous with the melibiose carrier of E . coli (23% similarity overall; greater than 50% similarity for regions with at least 16 amino acids), whereas the COOH-terminal end showed 34 to 41% similarity with the enzyme III (domain) of three different phosphoenolpyruvate-dependent phosphotransferase systems . Among the conserved amino acids were two histidyl residues, of which one has been postulated to be phosphorylated by HPr . Since sugars are not phosphorylated during translocation by the lactose transport system, it is suggested that the enzyme III-like region serves a regulatory function in this protein . The lacS gene also appears similar to the partially sequenced lactose transport gene of Lactobacillus bulgaricus (lacL; greater than 60% similarity) . Furthermore, the 3' flanking sequence of the S . thermophilus lactose transport gene showed approximately 50% similarity with the N-terminal portion of the beta-galactosidase gene of L . bulgaricus . In both organisms, the lactose transport gene and the beta-galactosidase appear to be separated by a 3-base-pair intercistronic region. Braz J Med Biol Res, 1989, 22(11), 1321 - 8 Restriction endonucleases from microorganisms isolated in Brazil: an isoschizomer of HaeIII from a thermophilic Bacillus sp; Cruz AK et al.; 1 . The isolation and characterization of a restriction endonuclease from a thermophilic strain of Bacillus is described . 2 . The enzyme recognizes the palindromic sequence 5'...GGCC...3' as determined by PEI-cellulose chromatography of pancreatic DNAse and snake venom phosphodiesterase digestion products of labelled DNA fragments, analysis of restriction digests and direct sequence analysis . 3 . The enzyme, denominated BspBR, is an isoschizomer of HaeIII and BspRI. J Basic Microbiol, 1989, 29(8), 491 - 9 Fungal flora of poultry feedstuff ingredients; Moharram AM et al.; One hundred and ten samples representing five types of poultry feed ingredients were mycologically examined . These samples included soybean meal, ground maize, cotton-seed cake, wheat bran and fish meal (22 samples each) . Among the 73 mesophilic fungal species, Aspergillus flavus, A . niger and A . fumigatus were the most dominant . A . terreus, A . flavipes, Mucor circinelloides, Scopulariopsis brevicaulis, Penicillium chrysogenum, Fusarium moniliforme and Rhizopus stolonifer were found to be common on a particular ingredient and less common on the remainders . Of the twelve thermophilic and thermotolerant species, A . fumigatus, Thermomyces lanuginosus, Rhizomucor pusillus and Thermoascus thermophilus prevailed on one or more type of the different ingredients . Marked variations were observed in the rancid fatty compounds of the different samples and the values ranged between 0.663 and 3.900 mg malonaldehyde per kg sample. Comp Biochem Physiol C, 1989, 92(1), 139 - 42 Phagocytosis in Tetrahymena thermophila: naloxone-reversible inhibition by opiates; De Jesus S et al.; 1 . Nanomolar concentrations of opiates inhibit phagocytosis in the ciliated protozoan Tetrahymena thermophila . 2 . Naloxone and naltrexone counteract the effect of the opiate agonists tested . 3 . The dose-response curves are U-shaped, with no detectable effect at low or high concentrations . 4 . An increase in extracellular calcium and dopamine counteract the inhibition caused by metenkephalin . 5 . The recognition mechanism for opiates in Tetrahymena cannot be classified as belonging to any of the mammalian opiate receptor subtypes and is perhaps a primitive receptor. Intervirology, 1989, 30(6), 323 - 9 Preliminary characterization of a group of actinophages of the thermophilic actinomycete genus Saccharomonospora; Schneider J et al.; The comparison of the Saccharomonospora phages phi SaC1, phi SaG1, phi SaV1, phi SaV2, phi SaV3, and Tm1 demonstrated that they all belong to a single group of closely related actinophages: they were similar with regard to host range, virion mophology (B1 type), genome length (43-45 kb), and GC content of their genomes (63, 3% G + C) . Furthermore, DNA-DNA hybridization showed that the phage genomes had a high degree of homology to each other . The phages are, therefore, proposed as representatives of a new bacteriophage species in the sense of the ICTV. J Bacteriol, 1989 Jan, 171(1), 65 - 9 Amino acid sequence of cytochrome c-552 from a thermophilic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus; Sanbongi Y et al.; The complete amino acid sequence of cytochrome c-552 from an extremely thermophilic hydrogen bacterium, Hydrogenobacter thermophilus TK-6 (IAM 12695), was determined . It is a single polypeptide chain of 80 residues, and its molecular weight, including heme, was calculated to be 7,599 . The sequence of cytochrome c-552 from H . thermophilus TK-6 closely resembles that of cytochromes c-551 from Pseudomonas species . Moreover, the tertiary structure of Hydrogenobacter cytochrome c-552 is suggested to be similar to that of cytochrome c-551 from Pseudomonas aeruginosa . The sequence similarity between Hydrogenobacter cytochrome c-552 and that of other bacteria physiologically related to H . thermophilus is not high. Int J Biochem, 1989, 21(11), 1203 - 10 Isocitrate dehydrogenase from thermophilic and mesophilic bacteria . Isolation and some characteristics; Edlin JD et al.; 1 . Simple methods incorporating the principle of selective enzyme elution from a triazinyl dye adsorbent with a mixture of NADP+ and isocitrate are described for isolating NADP+-linked isocitrate dehydrogenase in pure state from several mesophilic and thermophilic bacteria . 2 . Several characteristics of the isocitrate dehydrogenases have been examined, viz . molecular size, amino acid composition including the content of sulphydryl groups, thermostability and structural homology by the criterion of immunological cross-section. Arch Microbiol, 1989, 151(2), 159 - 65 A new bacteriocinogenic activity: megacin BII encoded by plasmid pSE 203 in strains of Bacillus megaterium; Stahl S; Mesophilic strains producing a new bacteriocin: Megacin BII, have been isolated from strains of Bacillus megaterium . Facultatively thermophilic strains producing Megacin BI were less sensitive to this new activity than non-producing mesophiles and strains producing Megacin BII were also more resistant to Megacin BI . Strains producing Megacin BII contained a large plasmid of 36.10(6):pSE 203 . This plasmid was introduced into non-megacinogenic acceptor strains by protoplast transformation, they then became megacin producers and immune to Megacin BII . Plasmid pSE 203 has been mapped with endonucleases . No similarity to the Megacin A plasmids pBM 309 {Rostas et al . (1980) and pBM 113 (von Tersch and Carlton (1983 b)} was evident. Nucleic Acids Symp Ser, 1989, (21), 117 - 8 Mg2+ effects on a circle opening reaction of the self-splicing intervening sequence from Tetrahymena thermophila; Sugimoto N et al.; The Mg2+ contribution to the reaction of circular intervening sequence (CIVS) from rRNA precursor of Tetrahymena thermophila with a dinucleotide CU has been investigated . The results indicated that the circle opening of CIVS may involve binding of a weakly held Mg2+ ion. Can J Microbiol, 1989 Jan, 35(1), 210 - 4 Comparison of transfer RNA and ribosomal RNA intron splicing in the extreme thermophile and archaebacterium Desulfurococcus mobilis; Kjems J et al.; The structure of the exon-intron boundary was compared for an intron within 23S ribosomal RNA of Desulfurococcus mobilis and a newly discovered intron in tRNA(Met) from the same organism . The occurrence of a putative common structural feature suggests that intron excision occurs by the same mechanism . The possible recognition of this structural feature by the cleavage enzyme was investigated for the ribosomal RNA intron using RNA substrates exhibiting various exon and intron deletions . The results support the involvement of the structural features in the cleavage process . The evolutionary implications of these results are considered. Biochemistry, 1988 Dec 27, 27(26), 9132 - 9 Affinity labeling of the GDP/GTP binding site in Thermus thermophilus elongation factor Tu; Peter ME et al.; Elongation factor Tu from Thermus thermophilus was treated successively with periodate-oxidized GDP or GTP and cyanoborohydride . Covalently modified cyanogen bromide or trypsin fragments of the protein were isolated, and the position of their modification was determined . Lysine residues 52 and 137 were heavily labeled, lysine-137 being considerably more reactive in the GTP form as compared to the GDP form of the protein . These residues are in the proximity of the GDP/GTP binding site . Lys-325 was also labeled, but to a lower extent . The part of the EF-Tu containing residue 52 is missing in crystallized EF-Tu.GDP from Escherichia coli {Jurnak, F . (1985) Science (Washington, D.C.) 230, 32-36} . These results place the part of T . thermophilus EF-Tu corresponding to the missing fragment in E . coli EF-Tu in the vicinity of the nucleotide binding site and allow its role in the interaction with aminoacyl-tRNA and elongation factor Ts to be evaluated . Cross-linking of EF-Tu.GDP by irradiation at 257 nm showed that a sequence of 10 amino acids residues which is found in the Thermus thermophilus elongation factor Tu but not in other homologous bacterial proteins is located in the vicinity of the GDP/GTP binding site. Biochim Biophys Acta, 1988 Dec 20, 951(2-3), 261 - 7 A DNA polymerase from a thermoacidophilic archaebacterium: evolutionary and technological interests; Elie C et al.; The archaebacteria constitute a group of prokaryotes with an intermediate phylogenetic position between eukaryotes and eubacteria . The study of their DNA polymerases may provide valuable information about putative evolutionary relationships between prokaryotic and eukaryotic DNA polymerases . As a first step towards this goal, we have purified to near homogeneity a DNA polymerase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius . This enzyme is a monomeric protein of 100 kDa which can catalyze DNA synthesis using either activated calf thymus DNA or oligonucleotide-primed single-stranded DNA as a template . The activity is optimal at 70 degrees C and the enzyme is thermostable up to 80 degrees C; however, it can still polymerize up to 200 nucleotides at 100 degrees C . These remarkable thermophilic properties and thermostability permit examination of the mechanism of DNA synthesis under conditions of decreased stability of the DNA helix . Furthermore, these properties make S . acidocaldarius DNA polymerase a very efficient enzyme to be used in DNA amplification by the recently developed polymerase chain reaction method (PCR) as well as in the Sanger DNA sequencing technique. Gene, 1988 Dec 20, 73(2), 273 - 94 Molecular genetics of group I introns: RNA structures and protein factors required for splicing--a review; Burke JM; In vivo and in vitro genetic techniques have been widely used to investigate the structure-function relationships and requirements for splicing of group-I introns . Analyses of group-I introns from extremely diverse genetic systems, including fungal mitochondria, protozoan nuclei, and bacteriophages, have yielded results which are complementary and highly consistent . In vivo genetic studies of fungal mitochondrial systems have served to identify cis-acting sequences within mitochondrial introns, and trans-acting protein products of mitochondrial and nuclear genes which are important for splicing, and to show that some mitochondrial introns are mobile genetic elements . In vitro genetic studies of the self-splicing intron within the Tetrahymena thermophila nuclear large ribosomal RNA precursor (Tetrahymena LSU intron) have been used to examine essential and nonessential RNA sequences and structures in RNA-catalyzed splicing . In vivo and in vitro genetic analysis of the intron within the bacteriophage T4 td gene has permitted the detailed examination of mutant phenotypes by analyzing splicing in vivo and self-splicing in vitro . The genetic studies combined with phylogenetic analysis of intron structure based on comparative nucleotide sequence data {Cech 73 (1988) 259-271} and with biochemical data obtained from in vitro splicing experiments have resulted in significant advances in understanding the biology and chemistry of group-I introns. J Cell Biol, 1988 Dec, 107(6 Pt 2), 2679 - 88 Fractionation of Tetrahymena ciliary membranes with triton X-114 and the identification of a ciliary membrane ATPase; Dentler WL; Cilia were isolated from Tetrahymena thermophila, extracted with Triton X-114, and the detergent-soluble membrane + matrix proteins separated into Triton X-114 aqueous and detergent phases . The aqueous phase polypeptides include a high molecular mass polypeptide previously identified as a membrane dynein, detergent-soluble alpha and beta tubulins, and numerous polypeptides distinct from those found in axonemes . Integral membrane proteins partition into the detergent phase and include two major polypeptides of 58 and 50 kD, a 49-kD polypeptide, and 5 polypeptides in relatively minor amounts . The major detergent phase polypeptides are PAS-positive and are phosphorylated in vivo . A membrane-associated ATPase, distinct from the dynein-like protein, partitions into the Triton X-114 detergent phase and contains nearly 20% of the total ciliary ATPase activity . The ATPase requires Mg++ or Ca++ and is not inhibited by ouabain or vanadate . This procedure provides a gentle and rapid technique to separate integral membrane proteins from those that may be peripherally associated with the matrix or membrane. Curr Genet, 1988 Dec, 14(6), 617 - 26 DNA rearrangements associated with the H3 surface antigen gene of Tetrahymena thermophila that occur during macronuclear development; Tondravi MM; The surfaces of Tetrahymena thermophila cells grown between 20 and 35 degrees C are covered by one or more variants of H antigens . A cDNA clone, pC6, has previously been identified that hybridizes to a unique polyA+ RNA that appears to code for the SerH3 variant of the H antigens . pC6 and a subclone of it, pGpC6.295, were used to analyze the genomic organization of the corresponding gene(s) in both the macronucleus and the micronucleus . It was determined that pC6 hybridizes to a small family of sequences in the macronucleus, only one of which also hybridizes to pGpC6.295 . The latter is a strong candidate for the gene encoding the SerH3 antigen . Sequences homologous to pC6 - but not to pGpC6.295 - are present in strains carrying the other SerH alleles . Shifts in antigen switching during vegetative growth do not result in any detectable DNA rearrangements in the vicinity of the pC6-hybridizing sequence family . Analysis of micronuclear DNA from a homozygous SerH3 strain revealed that it also contains a family of sequences that are homologous to pC6; but, in contrast to the macronuclear DNA, two members of this micronuclear sequence family hybridize to pGpC6.295 . Comparison of micro- and macronuclear DNA indicate that some members of the pC6-positive sequence family rearrange during macronuclear development . These rearrangements fall into two classes: those which occur reproducibly, and those which show variability . The gene homologous to pGp6.295 falls into the former category. J Bacteriol, 1988 Dec, 170(12), 5848 - 54 Cloning and sequencing of the gene encoding thermophilic beta-amylase of Clostridium thermosulfurogenes; Kitamoto N et al.; A gene coding for thermophilic beta-amylase of Clostridium thermosulfurogenes was cloned into Bacillus subtilis, and its nucleotide sequence was determined . The nucleotide sequence suggested that the thermophilic beta-amylase is translated from monocistronic mRNA as a secretory precursor with a signal peptide of 32 amino acid residues . The deduced amino acid sequence of the mature beta-amylase contained 519 residues with a molecular weight of 57,167 . The amino acid sequence of the C . thermosulfurogenes beta-amylase showed 54, 32, and 32% homology with those of the Bacillus polymyxa, soybean, and barley beta-amylases, respectively . Twelve well-conserved regions were found among the amino acid sequences of the four beta-amylases . To elucidate the mechanism rendering the C . thermosulfurogenes beta-amylase thermophilic, its amino acid sequence was compared with that of the B . polymyxa beta-amylase . The C . thermosulfurogenes beta-amyulase contained more Cys residues and fewer hydrophilic amino acid residues than the B . polymyxa beta-amylase did . Several regions were found in the amino acid sequence of the C . thermosulfurogenes beta-amylase, where the hydrophobicity was remarkably high as compared with that of the corresponding regions of the B . polymyxa beta-amylase. Biochim Biophys Acta, 1988 Nov 10, 951(1), 149 - 56 Characterization of the chromosomal protein MC1 from the thermophilic archaebacterium Methanosarcina sp . CHTI 55 and its effect on the thermal stability of DNA; Chartier F et al.; In the deoxyribonucleoprotein complex of Methanosarcina sp . CHTI 55, DNA is associated with two proteins, named MC1 (methanogen chromosomal protein 1) (Mr 10,760) and MC2 (Mr 17,000) . Protein MC1, the most abundant of these proteins, is closely related to the Methanosarcina barkeri MS protein MC1 . The effect of Methanosarcina sp . CHTI 55 protein MC1 on the thermal stability of DNA has been studied in native deoxyribonucleoprotein complex, as well as in reconstituted complexes, and it has been compared to the effect of E . coli DNA-binding protein II . Both proteins are able to protect DNA against thermal denaturation, but the differences observed in the melting profiles suggest that they interact by different mechanisms . Moreover, our studies indicate that one molecule of protein MC1 protects eight base pairs of DNA. Biochem Int, 1988 Nov, 17(5), 895 - 906 Isolation, purification and regeneration of protoplasts from Sporotrichum thermophile conidiospores; Sharma VK et al.; Protoplasts of uniform size were prepared from mononucleated conidiospores of Sporotrichum thermophile . Conidia were preincubated in glucose yeast extract medium at 45 C for 4 h . The conidia were collected resuspended in buffer containing 0.6 M KCl (as stabilizer), and incubated with Novozyme SP249 and Cellulase CP at 37 C for 6 h . The protoplasts were separated from cell wall fragments and intact conidia by centrifugation over 50% sucrose . The purified protoplasts were regenerated in glucose yeast extract broth after 7 h of incubation at 45 C. Genetics, 1988 Nov, 120(3), 697 - 705 A conditional mutant having paralyzed cilia and a block in cytokinesis is rescued by cytoplasmic exchange in Tetrahymena thermophila; Pennock DG et al.; Nineteen mutants that are conditional for both the ability to regain motility following deciliation and the ability to grow were isolated . The mutations causing slow growth were placed into five complementation groups . None of the mutations appears to affect energy production as all mutants remained motile at the restrictive temperature . In three complementation groups protein synthesis and the levels of mRNA encoding alpha-tubulin or actin were largely unaffected at the restrictive temperature, consistent with the hypothesis that mutations in these three groups directly affect the assembly of functional cilia and growth . Complementation group 1 was chosen for further characterization . Both phenotypes were shown to be linked, suggesting they are caused by a single mutation . Group 1 mutants regenerated cilia at the restrictive temperature, but the cilia were nonmotile . This mutation also caused a block in cytokinesis at the restrictive temperature but did not affect nuclear divisions or DNA synthesis . The block in cell division was transiently rescued by wild-type cytoplasm exchanged when mutants were paired with wild-type cells during conjugation (round 1 of genomic exclusion) . Thus, at least one mutation has been isolated that affects assembly of some microtubule-based structures in Tetrahymena (cilia during regeneration) but not others (nuclei divide at 38 degrees), and the product of this gene is likely to play a role in both ciliary function and in cytokinesis. Biochem J, 1988 Nov 1, 255(3), 865 - 8 A cell-associated oligo-1,6-alpha-glucosidase from an extremely thermophilic anaerobic bacterium, Thermoanaerobium Tok6-B1; Plant AR et al.; Cell-associated oligo-1,6-alpha-glucosidase (EC 3.2.1.10) was isolated from Thermoanaerobium Tok6-B1 grown on starch-containing medium . Activity was purified 11.4-fold by salt precipitation, gel filtration, hydroxyapatite and anion-exchange chromatography . Molecular mass was determined as 30,000 by SDS/polyacrylamide-gel electrophoresis and 33,000 by analytical gel filtration . The probable order of specificity was p-nitrophenyl-alpha D-glucose greater than-isomaltose greater than-isomaltotriose greater than-panose greater than-nigerose and no activity was shown against malto-oligosaccharides, melezitose, melibiose, raffinose, cellobiose, sophorose, gentiobiose, lactose, pullulan, dextran or amylose . The optima for activity and stability were between pH 5.6 and 7.0 and the half-life at pH 6.5 was 1000 min at 70 degrees C and 20 min at 76 degrees C . Activity was stabilized by substrate, Mg2+, Mn2+ and Ca2+, but was destabilized by Zn2+ and EDTA . N-Ethylmaleimide, glucose and 1-O-methyl-alpha D-glucose were inhibitory but 1-O-methyl-beta D-glucose stimulated activity . The activation energy (Ea) was 109 kJ/mol. Mol Cell Biol, 1988 Nov, 8(11), 5043 - 6 Reproducible and variable rearrangements of a Tetrahymena thermophila surface protein gene family occur during macronuclear development; Kile JP et al.; The expression of Tetrahymena surface proteins serotype H3 (SerH3) and serotype T (SerT) is under environmental regulation . SerH3 is expressed when cells are incubated between the temperatures of 20 and 35 degrees C, while SerT is expressed when cells are grown at temperatures above 35 degrees C . Using a SerH3 cDNA clone as a hybridization probe, we determined that (i) the SerH3 gene is a member of a multigene family; (ii) most members of this multigene family are variably rearranged during macronuclear development; and (iii) the gene which produces the SerH3 mRNA is reproducibly rearranged during macronuclear development. Mol Cell Biol, 1988 Nov, 8(11), 4780 - 6 Localization and expression of mRNA for a macronuclear-specific histone H2A variant (hv1) during the cell cycle and conjugation of Tetrahymena thermophila; White EM et al.; hv1 is a histone H2A variant found in the transcriptionally active Tetrahymena macronucleus but not in the transcriptionally inert micronucleus . This, along with a number of other lines of evidence, suggests that hv1 is associated with active genes . We have used a cDNA clone as a probe to study hv1 mRNA accumulation throughout the cell cycle and during conjugation . In situ hybridization to glutaraldehyde-fixed growing cells, whose position in the cell cycle was determined by size and morphology, showed that hv1 message is present throughout the cell cycle . The message was uniformly distributed in these vegetative cells . Compared with four other Tetrahymena histone genes studied to date (S . -M . Yu, S . Horowitz, and M . A . Gorovsky, Genes Dev., 1:683, 1987; M . Wu, C . D . Allis, and M . A . Gorovsky, Proc . Natl . Acad . Sci . USA 85:2205, 1988), hv1 mRNA is the only one that does not show a pattern of accumulation during the cell cycle that could explain the nuclear localization of its encoded protein . Thus, either hv1 or some molecule with which it associates contains a macronuclear-specific targeting sequence or there exists a cell cycle-regulated event that restricts its translation to the macronuclear S phase . In situ hybridization to conjugating cells revealed that hv1 message amounts increase just prior to macronuclear development and decline precipitously after the cells separate . The hv1 message showed no marked subcellular localization and is, therefore, unlikely to play a role in the cytoplasmic determination known to occur during macronuclear development. EMBO J, 1988 Nov, 7(11), 3531 - 7 Two guanosine binding sites exist in group I self-splicing IVS RNAs; Kay PS et al.; A shortened form of the self-splicing rRNA intervening sequence (IVS) of Tetrahymena thermophila can catalyze a transesterification reaction, termed G-exchange, between a monomeric guanosine derivative such as GTP and the substrate GpN (where N is A, C, G or U) . The reaction is specific to the two guanosines involved, providing evidence that two guanosine binding sites exist in this group I IVS RNA . One binding site accommodates a guanosine which initiates self-splicing and the other recognizes the guanosine preceding the 3' splice site . Previously, only one guanosine binding site was thought to be involved in the mechanism of self-splicing . Based on the two functionally distinguishable guanosine binding sites, a new model is proposed to explain how the two independent transesterification reactions required for self-splicing might proceed in a concerted manner. Eur J Biochem, 1988 Nov 1, 177(2), 273 - 80 S-adenosylmethionine synthetase in the thermophilic archaebacterium Sulfolobus solfataricus . Purification and characterization of two isoforms; Porcelli M et al.; Two isoforms of methionine adenosyltransferase (S-adenosylmethionine synthetase), A and B, have been partially purified from Sulfolobus solfataricus, a thermophilic archaebacterium optimally growing at 87 degrees C . The chromatographic procedure, involving hydrophobic chromatography on a phenyl-Sepharose column as a major step, results in 330-fold and 150-fold purification of adenosylmethionine synthetase A and B respectively . The apparent molecular masses, estimated by gel filtration, are 180 kDa for A and 75 kDa for B . The A and B isoforms follow Michaelis-Menten kinetics with apparent Km values of 10 microM and 20 microM for L-methionine and of 50 microM and 150 microM for ATP respectively . Adenosylmethionine, a product of the reaction, acts as a powerful non-competitive inhibitor (Ki = 50 microM) of the A isoform while it inhibits only slightly the B isoform . Both isozymes exhibit tripolyphosphatase activity but only that associated with the form A is stimulated by 5 microM adenosylmethionine concentration . The two enzymes absolutely require a divalent cation for the activity, but are not affected by monovalent ions and reducing agents . The optimum temperature is 90 degrees C and no significant loss of activity is observable after incubation of the two isoforms at 100 degrees C in the presence of ATP . The Arrhenius plots observed for both isozymes are biphasic, indicating different activation energies below and above 75 degrees C . The cytoplasmic levels of ATP, methionine and adenosylmethionine are evaluated. J Biochem (Tokyo), 1988 Nov, 104(5), 679 - 80 Crystallization and preliminary X-ray data for 3-isopropylmalate dehydrogenase of Thermus thermophilus; Katsube Y et al.; The gene coding for 3-isopropylmalate dehydrogenase of Thermus thermophilus was cloned and expressed in Escherichia coli . The extracted enzyme was crystallized in a suitable size for X-ray crystallographic studies . The crystals have a space group of P3(1)21 or P3(2)21 with a = b = 78.6 A and c = 157.4 A. J Biol Chem, 1988 Oct 25, 263(30), 15444 - 8 Purification and characterization of acetate kinase from acetate-grown Methanosarcina thermophila . Evidence for regulation of synthesis; Aceti DJ et al.; Acetate kinase was purified 102-fold to a specific activity of 656 mumol of ADP formed/min/mg of protein from acetate-grown Methanosarcina thermophila . The enzyme was not intrinsically membrane bound . The native enzyme (Mr 94,000) was an alpha 2 homodimer with a subunit Mr of 53,000 . The activity was optimum between pH 7.0 and 7.4 . A pI of 4.7 was determined . The enzyme was stable to O2 and stable to heating at 70 degrees C for 15 min but was rapidly inactivated at higher temperatures . The apparent Km for acetate was 22 mM and for ATP was 2.8 mM . The enzyme phosphorylated propionate at 60% of the rate with acetate but was unable to use formate . TTP, ITP, UTP, GTP, and CTP replaced ATP as the phosphoryl donor to acetate . The enzyme required one of several divalent cations for activity; the maximum rate was obtained with Mn2+ . Western blots of cell extract proteins showed that acetate grown cells synthesized higher quantities of the acetate kinase than did methanol grown cells. Biochem J, 1988 Oct 15, 255(2), 699 - 703 A DNA-modification methylase from Bacillus stearothermophilus V; Barra R et al.; A type II modification methylase (M BstVI) was partially purified from the thermophilic bacterium Bacillus stearothermophilus V . The methylase catalyses the transfer of methyl groups from S-adenosyl-L-methionine to unmodified double-stranded DNA . The product of methylation was identified by paper chromatography as N6-methyladenine . Since M BstVI protects DNA against cleavage by BstVI and XhoI restriction endonucleases, it follows that it methylates the adenine residue in the sequence 5'-C-T-C-G-A-G-3'. J Mol Biol, 1988 Oct 5, 203(3), 831 - 4 Characterization of crystals of small ribosomal subunits; Yonath A et al.; Crystals of intact small ribosomal subunits from Thermus thermophilus have been obtained from functionally active particles . The crystals (P42(1)2, 407 A x 407 A x 171 A) are suitable for X-ray crystallography analysis to 9.9 A using synchrotron radiation at cryotemperature . Crystallographic data from native and a potential heavy-atom derivative have been collected. J Biochem (Tokyo), 1988 Oct, 104(4), 557 - 9 Determination of the positions of the disulfide bonds in aqualysin I (a thermophilic alkaline serine protease) of Thermus aquaticus YT-1; Kwon ST et al.; Aqualysin I is a heat-stable alkaline serine protease produced by Thermus aquaticus YT-1 . Aqualysin I comprises 281 amino acid residues and contains four cysteine residues . The cysteine residues seemed to form disulfide bonds in the molecule . Thus, the positions of the disulfide bonds were investigated . Disulfide bond-containing peptides were identified by peptide mapping with HPLC before and after carboxymethylation of chymotryptic peptides of aqualysin I . The disulfide bond-containing peptides were isolated and then carboxymethylated . Carboxymethylcysteine-containing peptides were purified, and their amino acid compositions and sequences were determined . Based on the data obtained and the primary structure of aqualysin I, it was concluded that two disulfide bonds were formed between Cys67 and Cys99, and between Cys163 and Cys194. Appl Environ Microbiol, 1988 Oct, 54(10), 2335 - 41 Effect of anaerobic digestion on oocysts of the protozoan Eimeria tenella; Lee MR et al.; The effect of anaerobic digestion of poultry waste on oocysts of the protozoan Eimeria tenella, a common enteric pathogen that causes coccidiosis in poultry, was investigated in this study . Thermophilic (50 degrees C) and mesophilic (35 degrees C) anaerobic digestors, with poultry manure as the substrate, were inoculated with the oocysts . The oocysts were damaged during anaerobic digestion, as determined by morphological change and loss of their ability to sporulate . The recovered oocysts were tested for their infectivity in young chicks, as measured by body weight gain, mortality, and cecal lesions . Oocysts lost all their infectivity during thermophilic digestion, while oocysts subjected to mesophilic digestion remained moderately infective in comparison with untreated oocysts, which produced severe coccidiosis, high mortality, and low body weight gain in chicks . Oocysts were inactivated at 50 degrees C when they were suspended in digestor fluid or saline . Inactivation at 35 degrees C was significantly stronger in the digestor fluid than in the saline, which implied that factors other than temperature were involved in the lethal effect of anaerobic digestion on protozoan oocysts . In this study we demonstrated that the treatment of an