Genomic characterization of thermophilic Geobacillus species isolated from the deepest sea mud of the Mariana Trench

The thermophilic strains HTA426 and HTA462 isolated from the Mariana Trench were identified as Geobacillus kaustophilus and G. stearothermophilus, respectively, based on physiologic and phylogenetic analyses using 16S rDNA sequences and DNA–DNA relatedness. The genome size of HTA426 and HTA462 was estimated at 3.23–3.49 Mb and 3.7–4.49 Mb, respectively. The nucleotide sequences of three independent -phage inserts of G. stearothermophilus HTA462 have been determined. The organization of protein coding sequences (CDSs) in the two -phage inserts was found to differ from that in the contigs corresponding to each insert assembled by the shotgun clones of the G. kaustophilus HTA426 genome, although the CDS organization in another insert is identical to that in the HTA426 genome.     

A mutant of Escherichia coli K12 deficient in the 5''-3'' exonucleolytic activity of DNA polymerase I. II. Purification and properties of the mutant enzyme

Summary The enzymatic properties of purified DNA polymerase I from a strain of Escherichia coli K12 with a mutation in the polA gene have been studied. The polymerizing activity of the mutant enzyme is similar to that of the enzyme from isogenic wild-type cells, when the activity is measured on exonuclease III treated calf-thymus DNA. Also the 3–5 exonucleolytic activity is not significantly different for both enzyme preparations. The 5–3 exonucleolytic activity of DNA polymerase I isolated from the mutant strain, however, is much lower than that of wild-type DNA polymerase I. The products formed by the action of the wild-type and the mutant enzyme on nicked circular double-stranded DNA of phage X174 (RFII DNA) were analysed by sucrose-gradient sedimentation and electron-microscopy. When RFII DNA was incubated with wild-type enzyme 80% of the molecules were converted into linear molecules. All linear molecules were shorter than one phage genome. Only 25% of the molecules were branched. After incubation of RFII DNA with the mutant enzyme 62% of the molecules have become linear. More than 90% of these linear molecules were branched and the majority of them was longer than one phage genome.     

Selection and analysis of non-interactive mutants in the Escherichia coli tryptophan synthase alpha subunit.

Summary The inherent infidelity of Taq DNA polymerase in the polymerase chain reaction was exploited to produce random mutations in thetrp A gene. Screening of the resulting clones allowed selection of non-interactive mutant subunits retaining their intrinsic catalytic activity. Two single changes responsible for this phenotype were identified by DNA sequencing as: 126 valine (GTG)glutamic acid (GAG) and 128 valine (GTT)aspartic acid (GAT). Three single changes giving a non-interactive phenotype with an impaired intrinsic catalytic activity were identified by DNA sequencing as a66 asparagine (AAC)aspartic acid (GAC); 109lysine (AAA) arginine (AGA); 118 cysteine (TGC)arginine (CGC). Where possible, we individually assessed the importance of these residues in interaction in light of structural information from X-ray crystallography and by intergeneric protein sequence comparison.     

Specific in vitro phosphorylation of plant eIF2α by eukaryotic eIF2α kinases

Phosphorylation of the subunit of eukaryotic initiation factor 2 (eIF2) is known to be an important translational control mechanism in all eukaryotes with the major exception of plants. Regulation of mammalian and yeast eIF2 activity is directly governed by specific phosphorylation on Ser-51. We now demonstrate that recombinant wheat wild-type (51S) but not mutant 51-Ala (51A) protein is phosphorylated by human PKR and yeast GCN2, which are defined eIF2 kinases. Further, only wheat wild-type eIF2 is a substrate for plant-encoded, double-stranded RNA-dependent kinase (pPKR) activity. Plant PKR and GCN2 phosphorylate recombinant yeast eIF2 51S but not the 51A mutant demonstrating that pPKR has recognition site capability similar to established eIF2 kinases. A truncated version of wild-type wheat eIF2 containing 51S but not the KGYID motif is not phosphorylated by either hPKR or pPKR suggesting that this putative eIF2 kinase docking domain is essential for phosphorylation. Taken together, these results demonstrate the homology among eukaryotic eIF2 species and eIF2 kinases and support the presence of a plant eIF2 phosphorylation pathway.     

Electrophoretic karyotyping from Tolypocladium inflatum and six related strains allows differentiation of morpologically similar species

Summary The electropheretic karyotype for the imperfect filamentous fungus Tolypocladium inflatum and for six related strains is presented. Pulsed-field gel electrophoresis was used with improved separation conditions to separate DNA from 6.6 Mb to 1.05 Mb in size. Using probes encoding rRNA or the -tubulin gene from T.iinflatum the corresponding genes were detected on designated chromosomes. In addition, two recombinant lambda clones, carrying T. inflatum chromosomal DNA, were used as chromosome-specific probes. Although all strains investigated are very similar in their morphology, significant chromosome-lengt polymorphisms were detected, allowing easy strain differentiation. The polymorphism was confirmed using an rRNA probe for genomic mapping. All strains contain a homologous minichromosome of 1.05 Mb. Finally, the resolution of very large DNA enabled us to separate a 6.6-Mb DNA band that specifically hybridizes with mitochondrial gene probes. The electrophoretic karyotyping presented here may be regarded as a reliable molecular tool to differentiate morphologically very similar filamentous fungi. Correspondence to: U. Kück     

Genome size and chromatin condensation in vertebrates

Cell membrane-dependent chromatin condensation was studied by flow cytometry in erythrocytes of 36 species from six classes of vertebrates. A positive relationship was found between the degree of condensation and genome size. The distribution of variances among taxonomic levels is similar for both parameters. However, chromatin condensation varied relatively more at the lower taxonomic levels, which suggests that the degree of DNA packaging might serve for fine-tuning the skeletal and/or buffering function of noncoding DNA (although the range of this fine-tuning is smaller than the range of genome size changes). For two closely related amphibian species differing in genome size, change in chromatin condensation under the action of elevated extracellular salinity was investigated. Condensation was steadier and its reaction to changes in solvent composition was more inertial in the species with a larger genome, which is in agreement with the buffering function postulated for redundant DNA. The uppermost genome size in vertebrates (and in living beings in general) was updated using flow cytometry and was found to be about 80 pg (78,400 Mb). The widespread opinion that the largest genome occurs in unicellular organisms is rejected as being based on artifacts.     

Inactivation of DNA polymerases by adenosine 2′,3′-riboepoxide 5′-triphosphate allows estimation of the primers affinity

Template-primer dependent inactivation of human DNA polymerase and Klenow fragment of E. coli DNA polymerase I by adenosine 2,3-riboepoxide 5-triphosphate was used for quantitative analysis of the K_d values for oligonucleotide primers of different length. The K_d values are smaller by a factor of 2.5 than the K_m values for the same primers determined in the reaction of DNA polymerization in the case of DNA polymerase . The K_d and K_m values are nearly the same for Klenow fragment. Such approach to the determination of K_m/K_d ratio can likely be used for detailed quantitative analysis of DNA polymerases.Abbreviations epATP adenosine 2,3-riboepoxide 5-triphosphate - KF Klenow fragment of E. coli DNA polymerase I - Pol I E. coli DNA polymerase I - Pol human placenta DNA polymerase      

Genetic polymorphism at the κ chain locus in mice: Comparisons of restriction enzyme hybridization fragments of variable and constant region genes

Variable (V) and constant (C) region genes of the mouse kappa light chain have been compared in inbred strains and in geographically isolated or genetically separated populations of mice by Southern blot analysis of endonuclease-restricted germline DNA. In most cases, the C gene is found on a single restriction fragment while the V genes of the V19 and V21 groups are each found on several (6–18) fragments. The restriction fragment (RF) patterns of V19 and V21 groups are both polymorphic when compared among inbred mouse strains. Southern blot patterns of V21 and V19 of inbred strains are also found among some geographically isolated populations of mice, suggesting that inbred strains acquired kappa loci from different subspecies. Some populations of geographical isolates show V21, V19, and C contexts similar to inbred mice while more distantly related species within the genus Mus and laboratory rats show no apparent similarity in context to inbred strains. Variable region genes determining the RF patterns of V19 and V21 appear to be linked to each other and to the C and Lyt-3 loci.     

Complementation analysis with new genetic markers in Phaffia rhodozyma

Isolation and characterization of auxotrophic mutants from wild-type and astaxanthin mutant strains of Phaffia rhodozyma is described. Differences in survival were observed when u.v. irradiation of P. rhodozyma wild-type and astaxanthin mutant strains were incubated in the dark or exposed to photoreactivating light. Ultra-violet mutagenesis was not effective to produce auxotrophic mutants in this yeast. Auxotrophic mutants were obtained with high efficiency through a nystatin enrichment procedure after a N-methyl-N-nitro-N-nitrosoguanidine (NTG) mutagenic treatment with a 0.12% survivor level. Stringent mutagenetic conditions were needed to obtain P. rhodozyma auxotrophs. The most frequent mutants were ade^- and met^- in a rather narrow auxotroph spectrum. These results may be associated with a possible diploid condition of this yeast. The high number of adenine auxotrophs obtained in relation to other auxotrophic mutants suggests the possibility of some degree of heterozygosity in the wild-type strain UCD 67-385.     

Gene organization and plasticity of the β-lactam genes in different filamentous fungi

The genes pcbAB, pcbC and penDE encoding enzymes that catalyze the three steps of the penicillin biosynthesis have been cloned from Penicillium chrysogenum and Aspergillus nidulans. They are located in a cluster in Penicillium chrysogenum, Penicillium notatum, Aspergillus nidulans and Penicillium nalgiovense. The three genes are clustered in chromosome I (10.4 Mb) of P. chrysogenum, in chromosome II of P. notatum (9.6 Mb) and in chromosome VI (3.0 Mb) of A. nidulans. The cluster of the penicillin biosynthetic genes is amplified in strains with high level of antibiotic production. About five to six copies of the cluster are present in the AS-P-78 strain and 11 to 14 copies in the E1 strain (an industrial isolate), whereas only one copy is present in the wild type (NRRL 1951) strain and in the low producer Wis 54-1255 strain. The amplified region in strains AS-P-78 and E1 is arranged in tandem repeats of 106.5 or 57.6-kb units, respectively. In Acremonium chrysogenum the genes involved in cephalosporin biosynthesis are separated in at least two clusters. The pcbAB and pcbC genes are linked in the so-called early cluster of genes involved in the cephalosporin biosynthesis. The late cluster, which includes the cefEF and cefG genes, is involved in the last steps of cephalosporin biosynthesis. The early cluster was located in chromosome VII (4.6 Mb) in the C10 strain and the late cluster in chromosome I (2.2 Mb). Both clusters are present in a single copy in the A. chrysogenum genome, in the wild-type and in the high cephalosporin-producing C10 strains.     

Saccharomyces cerevisiae 2-mum DNA. An analysis of the monomer and its multimers by electron microscopy.

Summary The non-tandem inverted duplication in the 2-m DNA of Saccharomyces cerevisiae has a length of 0.19 m and is located asymmetrically along the molecule. The majority of the dumb-bell structures that are formed upon denaturation and selfannealing of the 2-m monomer consists of the renatured inverted duplication sequences as double stranded stem and two single stranded loops of 0.67 m±0.06 m (S-loop) and 0.86 m±0.05 m (L-loop) length. Two additional size classes which comprised 5–10% of the measured molecules had contour lengths of around 1.7 m and 2.1 m. The smaller dumb-bells contained two S-loops and the larger dumb-bells contained two L-loops as was shown by heteroduplex mapping with an HindIII fragment from the L-loop. Two models which assume illegitimate or site specific recombination, are presented to explain the generation of double S-loop and double L-loop molecules. At least part of the 4-m and 6- circular molecules present in the yeast supercoiled DNA fraction are shown to be dimers and trimers of 2-m monomers, but often with inverted loop segments most probably due to intramolecular recombination between sequences of the inverted duplication.2-m DNA is used to indicate the supercoiled DNA fraction although in our measurements the average monomeric length is 1.9 mPart of this work has been presented at the Conference: The Genetics and Biogenesis of Chloroplasts and Mitochondria, Munich, August, 1976     

Molecular cloning of Bacillus subtilis genes involved in DNA metabolism

Summary Different clones carrying a chromosomal DNA fragment able to transform Bacillus subtilis mutants dnaA13, dnaB19, dnaG5, recG40 and polA42 to a wild-type phenotype were isolated from a library constructed in plasmid pJH101. A recombinant clone carrying a chromosomal fragment able to transform dnaC mutants was obtained from a Charon 4A library. A restriction map of the cloned DNA fragments was constructed. The 11.3 kb cloned DNA fragment of plasmid pMP60-13 containing the wild-type sequence of dnaG5 was shown to transform a recF33 mutant as well.     

Upper limit for DNA packaging by Bacillus subtilis bacteriophage phi 105: isolation of phage deletion mutants by induction of oversized prophages

Summary We have determined the upper size limit for DNA packaging in Bacillus subtilis bacteriophage 105 by examining the plaque-forming and transducing capabilities of lysates made from strains containing prophages of various sizes. The upper size limit for efficient packaging of the phage genome appears to be about 40.2 kb, which is about 1 kb larger than the wild-type genome. This places an upper limit of about 5 kb on the size of insertions that can be accommodated in 105 transfection cloning vectors, such as 105J27. Induction of prophages that exceed the upper limit, followed by selection for plaque formation or transduction, provides a powerful means of isolating phage deletion mutants. A comparison of the location of each deletion with the resultant phenotype has enabled us to identify non-essential regions of the phage genome, and regions that are required for tail biosynthesis and for host cell lysis.     

Transposome Insertional Mutagenesis and Direct Sequencing of Microbial Genomes

Preformed transposase-transposon complexes called Transposomes have been electroporated into bacterial cells. The magnesium dependent process of insertion of the transposable element into bacterial chromosomal DNA occurs in vivo. The transposition efficiency of a Transposome containing a kanamycin marker was between 1.0×10⁴and 1.0×10⁷kanamycin resistant clones per microgram of transposon DNA in three gram-negative enteric bacterial species. Transposon integration sites were examined by direct genome sequencing of chromosomal DNA. Genomic DNA was isolated from transposition clones and directly cycle sequenced with primers specific for the ends of the transposon. The precise location of genome interruption for a transposition clone was identified by homology to known genes or sequences. Mutant phenotypes were rapidly correlated with genomic insertions sites.     

Electrophoretic karyotypes of Phaffia rhodozyma strains

Abstract Electrophoretic karyotypes of strains from the astaxanthin-producing yeast Phaffia rhodozyma have been established. Intact chromosomal DNA molecules released from protoplasts were separated by orthogonal field alternation gel electrophoresis (OFAGE) and contour clamped homogeneous electric field (CHEF). Both small and large chromosomal DNA molecules were resolved simultaneously by optimizing the running conditions. Electrophoretic karyotypes among the Phaffia isolates examined differed significantly. Seven to thirteen chromosomal bands, ranging in size from 0.83 Mb to 3.50 Mb, were resolved, giving total genome sizes of about 15.4 to 23.2 Mb. Ribosomal DNA has been assigned to chromosomal bands using a heterologous gene probe.     

Interleukin-1 α and β genes: linkage on chromosome 2 in the mouse

Two interleukin-1 polypeptides, and , are known, and cDNAs corresponding to each have been described. Genomic cloning and Southern blotting experiments suggest that in the mouse each is encoded by a gene present in one copy per haploid genome. Analysis of a panel of somatic cell hybrids carrying various mouse chromosomes on a constant Chinese hamster background indicates that both genes map to mouse chromosome 2. Further, analysis of the inheritance of DNA restriction fragment length polymorphisms associated with each gene in recombinant inbred strains of mice shows the two loci to be tightly linked to one another, and to lie approximately 4.7 centimorgans distal to B2m (beta-2 microglobulin). We have named the locus encoding IL-1 Il-1 and the locus encoding IL-1 Il-1b.     

Nucleic acid metabolism in yeast

Summary The three haploid yeast strains T2tmp1-3, T2tmp1-1, and T6tmp1-51 auxotrophic for 5-dTMP differ in their requirement for thymidylate: 72, 16, and 3 g 5-dTMP/ml will restore optimal growth, respectively. Thymidylate low requirement in strain T2tmp1-1 and T6tmp1-51 is termed tlrA and tlrC, respectively. When the growth medium is made 5x10^-4 M for 5-dTMP only strain T6tmp1-51 is severely inhibited in RNA and DNA synthesis. This inhibition is reversible after removal of excessive 5-dTMP. The inhibitory characteristic is in marked contrast to thymineless death due to the lack of 5-dTMP in strain T6tmp1-51 where only DNA synthesis stops while RNA synthesis continues. The inhibitory effect of 5x10^-4 M 5-dTMP is not due to the 5-dTMP auxotrophy but to the thymidylate low requiring character (tlrC) in strain T6tmp1-51. The arrest of RNA and DNA synthesis by high concentrations of exogenous 5-dTMP suggests a regulatory role of either the monoor triphosphate on nucleoside or nucleotide biosynthesis in yeast.     

The starting point and direction of rolling-circle replicative intermediates of coliphage lambda DNA.

Summary Intermediates of DNA replication in the second half of the latent period after phage infection were isolated and investigated in the electron microscope by denaturation mapping. The isolated replicative froms (RF) are predominantly single branched circular DNA. The starting points of replication in these lariat molecules located at the same region as in the first round DNA replication. About 60% of the RF replicate from left to right and the other 40% replicate in the reverse direction. The free ends of the tails are located at many sites on the genome. Replicating circles with a linear DNA tail longer than one unit length of genome represent about 30% of the replicating molecules. These long linear tails (concatemers) produced by the rolling-circle (Gilbert and Dressler, 1968; Eisen et al., 1968; Skalka et al., 1972; Takahashi, 1974) are one of the best candidates for a precursor DNA of progeny phage.