Complete Sequence and Gene Organization of the Genome of a Hyper-thermophilic Archaebacterium, Pyrococcus horikoshii OT3

The complete sequence of the genome of a hyper-thermophilicarchaebacterium, Pyrococcus horikoshii OT3, has been determinedby assembling the sequences of the physical map-based contigsof fosmid clones and of long polymerase chain reaction (PCR)products which were used for gap-filling. The entire lengthof the genome was 1,738,505 bp. The authenticity of the entiregenome sequence was supported by restriction analysis of longPCR products, which were directly amplified from the genomicDNA. As the potential protein-coding regions, a total of 2061open reading frames (ORFs) were assigned, and by similaritysearch against public databases, 406 (19.7%) were related togenes with putative function and 453 (22.0%) to the sequencesregistered but with unknown function. The remaining 1202 ORFs(58.3%) did not show any significant similarity to the sequencesin the databases. Sequence comparison among the assigned ORFsin the genome provided evidence that a considerable number ofORFs were generated by sequence duplication. By similarity search,11 ORFs were assumed to contain the intein elements. The RNAgenes identified were a single 16S-23S rRNA operon, two 5S rRNAgenes and 46 tRNA genes including two with the intron structure.All the assigned ORFs and RNA coding regions occupied 91.25%of the whole genome. The data presented in this paper are availableon the internet at http://www.nite.go.jp.     

Characterization and Genetic Mapping of Simple Sequence Repeats in the Rice Genome

We searched partial sequences of over 22,706 rice cDNA and 1220genomic DNA clones to find and characterize simple sequencerepeats (SSRs) in the rice genome. The most frequently foundrepeated SSR motif in both cDNA and genomic DNA sequences wasd(CCG/CGG)n. The second most frequently found SSR was d(AG/CT)n.In contrast with mammalian genomes, in which d(AC/GT)n sequencesare the most abundant, d(AC/GT)n sequences were not frequentlyobserved in rice. Sequences containing d(CCG/CGG)n, d(AG/CT)nrepeats, and other SSRs were chosen for polymorphism detection.It was predicted that 17 of 20 SSRs in cDNA sequences were locatedin 5'-untranslated regions near initiation codons. Twenty-twoloci can be mapped on our RFLP linkage map by these SSRs. Sixmarkers were tested with 16 japonica rice varieties as templatesfor PCR. Two markers exhibited amplified fragment length polymorphismamong these rice varieties, implying that SSRs are polymorphicamong rice varieties which have similar genetic backgrounds.Even these polymorphic SSRs are located within or around geneswhich code ubiquitous proteins.     

Inhibitory effect of thyroid hormones on pituitary cyclic AMP phosphodiesterase activity in vitro

Thyroid hormones effectively inhibited partially purified cyclic AMP phosphodiesterase from the anterior and posterior lobes of bovine pituitary gland competitively with the substrate, but thyronine, diiodotyrosine, monoiodotyrosine, thyrotropin, thyrotropin releasing hormone, dexamethasone, and luteinizing hormone releasing hormone did not inhibit this enzyme activity.K _i values were found to be 1.0 and 2.5 M for thyroxine, and 8.0 and 13.5 M for triiodothyronine in the anterior and posterior lobes, respectively.     

Decreased Na+-gradient-dependent d-glucose transport in brush-border membrane vesicles from rabbits with experimental fanconi syndrome

The effect of anhydro-4-epitetracycline on sodium gradient-dependent d-glucose transport of rabbit renal brush-border membrane vesicles was studied. The purity of isolated brush-border membrane vesicles as judged by enzyme activities was not different between normal control and anhydro-4-epitetracycline-administered rabbits. There was no difference in estimate of intravesicular volume, either. When NaCl was used for sodium gradient, the overshoot of d-glucose uptake into brush-border membrane vesicles isolated from anhydro-4-epitetracycline-treated rabbits was significantly smaller than that of normal control rabbits. In the cases of NaSCN or Na₂SO₄, the former was also smaller than the latter, but not significantly so. To avoid the possible effect of membrane potential on d-glucose uptake, the voltage-clamp method was applied. Even in the voltage-clamped condition, the overshoot of d-glucose uptake into vesicles from anhydro-4-epitetracycline-treated rabbits was decreased compared to that of normal rabbits. In vitro incubation of brush-border membrane vesicles with 20 mM anhydro-4-epitetracycline caused no alteration in sodium gradient-dependent d-glucose uptake. Our results demonstrate that there exists a disorder in sodium gradient-dependent d-glucose uptake of renal brush-border membrane in anhydro-4-epitetracycline-treated rabbits, and suggest that this disorder is one of the underlying mechanisms of experimental Fanconi syndrome.     

Effect of Methyl Substitution on Microbial Degradation of Linear Styrene Dimers by Two Soil Bacteria

The microbial degradation of 10 linear unsaturated dimers (I to IV) prepared from styrene and o-, m-, or p-methylstyrene was investigated with two soil bacteria, Alcaligenes sp. strain 559 and Pseudomonas sp. strain 419. The two strains decomposed styrene dimer I and all styrene-methylstyrene codimers II and III, but methylstyrene homodimers IV remained intact. The degradation rates of codimers II and III of o- and m-methylstyrenes were found to depend on both their structure and the strain used; i.e., Alcaligenes sp. strain 559 decomposed III faster than II, whereas the reverse order (II > III) was obtained with Pseudomonas sp. strain 419. In biodegradation by the former strain, the codimers were degraded faster in the presence of styrene dimer I than in its absence, but no such effect of dimer I was observed with the latter.     

Effect of Yersinia enterocolitica ST on cyclic guanosine 3',5'-monophosphate levels in mouse intestines and cultured cells

We determined the effect of heat-stable enterotoxin produced by Yersinia enterocolitica (Y. enterocolitica ST) on cyclic nucleotide levels in the intestines of 6-day-old mice and in cultured cell line cells. The concentration of cyclic guanosine 3',5'-monophosphate (cyclic GMP) in homogenates of the intestines increased four- to fivefold by 3 min after intragastric administration of 10 units of purified Y. enterocolitica ST. This increase continued for 60 min, and then the concentration of cyclic GMP fell toward the levels of the controls. On the other hand, fluid accumulation in the intestines was not evident until 60 min after administration of the toxin. Thus, the increase in intestinal cyclic GMP concentration preceded measurable fluid accumulation. The effect on both cyclic GMP levels and fluid accumulation was abolished by treatment of the ST with either alkali solution (pH 10.7) or 2-mercaptoethanol. Likewise, cyclic GMP levels in cultured cells (CCL-6, HeLa, L, and Mm-1 cells) increased dose-dependently by 10 min after incubation of the cells with the ST. Cyclic adenosine 3',5'-monophosphate levels in both intestines and cultured cells were not affected by the toxin.     

Chromosome banding in the genusPinus

Somatic chromosomes ofPinus nigra var.maritima (2n=24) were sequentially stained with DNA binding base-specific fluorochromes, chromomycin A₃ (CMA) and DAPI. Many CMA- and DAPI-bands appeared at intercalary and/or proximal regions of most chromosomes. These banding patterns reversely related. Individual chromosomes were easily identified using these fluorescent banding patterns.     

Two distinct subtypes of theHLA-DRw12 haplotypes in the Japanese population detected by nucleotide sequence analysis and oligonucleotide genotyping

We determined the DNA sequence of the enzymatically amplified second exon of theDRB1 gene of theDRw12 haplotypes derived from three Japanese donors and found two distinct subtypes of theDRw12 haplotype. The two subtypes, designatedDRw12a andDrw12b, had single-base substitutions that predicted one amino acid change at residue number 67. The sequence of theDrw12a andDRw12b subtypes differed from those of the otherDR haplotypes, but in the first hypervariable region of theDRB1 gene the sequences were identical to those of theDRw8(Dw8.1) andDRw8(Dw8.3) haplotypes. TheDRw12a andDRw12b subtypes were detected in a wide range of Japanse donors by genotyping with sequence-specific oligonucleotide probes synthesized according to the DNA sequence of the two subtypes. Results of this study demonstrated that theDRw12 haplotypes in the Japanese population are genetically diverse, as many otherDR haplotypes are. The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers M27509, M27510, M27511.     

A Phanerochaete chrysosporium mutant defective in lignin degradation as well as several other secondary metabolic functions

A pleiotropic mutant of Phanerochaete chrysosporium 104-2 lacking phenol oxidase and unable to form fruit bodies and a revertant strain 424-2 were isolated after UV mutagenesis. Strains 104-2 and 424-2 had no apparent dysfunction in primary metabolism with glucose as a carbon source. Unlike the wild type strain and strain 424-2, strain 104-2 was unable to evolve ¹⁴CO₂ from ¹⁴C ring, side chain and 3-O-¹⁴C-methoxy labeled lignin. In addition, strain 104-2 was unable to evolve ¹⁴CO₂ from a variety of lignin model compounds including ¹⁴C-4-methoxy labeled veratrylglycerol--guaiacyl (V) ether, -¹⁴C-guaiacylglycerol--guaiacyl ether (VI), as well as 1-(¹⁴C-4-methoxy, 3-methoxyphenyl)1,2 propene (III) and 1-(¹⁴C-4-methoxy-3-methoxyphenyl) 1,2 dihydroxypropane (IV). The addition of peroxidase/H₂O₂ to cultures of strain 104-2 did not alter its capacity to degrade the labeled lignins. A variety of unlabeled lignin model compounds previously shown to be degraded by the wild type organism including -aryl ether dimers and diaryl propane dimers were also not degraded by the mutant 104-2. The revertant strain 424-2 regained the capacity to degrade these compounds. The substrates described are degraded by oxygen requiring system(s) expressed during the secondary phase of growth, suggesting this pleiotropic mutant is possibly defective in the onset of postprimary metabolism. The inability of the mutant to produce the secondary metabolite veratryl alcohol and to elaborate enzymes in the veratryl alcohol biosynthetic pathway supports this hypothesis.Abbreviations GLC gas liquid chromatography - TMSi trimethylsilyl - MS mass spectrometry - LDS lignin degrading system