Efficient Conversion of L-Tryptophan to Indole-3-Acetic Acid and/or Tryptophol by Some Species of Rhizoctonia

In a study of various phytopathogenic fungi, we found that fungithat belong to the genus Rhizoctonia produce IAA efficientlyfrom tryptophan. R. solani Kühn MAFF-305219, in particular,produced large amounts of tryptophol (Tol), which was assumedto be a specific by-product of the indole-3-pyruvate (IPy) pathway,in addition to IAA. Therefore, this fungus seemed suitable foranalysis of the function and the regulation of the biosynthesisof auxin by a fungal pathogen. Under normal aerobic conditions,the ratio of IAA to Tol synthesized by this strain was higherthan that under less aerobic conditions. In metabolic studieswith various indole derivatives, R. solani converted L-tryptophanand indole-3-acetaldehyde to IAA and Tol, but other indole derivativeswere scarcely metabolized. These results suggest that both IAAand Tol are synthesized from tryptophan through the IPy pathwayin Rhizoctonia. (Received May 27, 1996; Accepted July 8, 1996)     

Promotion of Cocklebur Seed Germination by Allyl, Sulfur and Cyanogenic Compounds

The effects of allyl, sulfur and cyanogenic compounds on thegermination of upper cocklebur (Xanthium pennsylvanicum Wallr.)seeds were examined. Mercaptoethanol and methylmercaptan aswell as KCN, substrates for rßcyanoalanine synthase(CAS), and H₂S and thiocyanate, the products of the CAS catalyzingreaction, were effective in promoting germination, suggestingthe involvement of CAS in germination. Most of allyl compounds, especially allylthiourea, as well asethylene which activated CAS [Hasegawa et al. (1994) Physiol.Plant. 91: 141], promoted the germination in an abnormal typewhich occurred by the predominant growth of cotyledons as didC₂H₄ [Katoh and Esashi (1975) Plant Cell Physiol. 16: 687].However, they failed to activate CAS unlike ethylene, and toliberate free ethylene during an incubation period. It was thuspossible that an C₂H₄-like double bond within allyl compoundscan act to promote seed germination. (Received June 10, 1996; Accepted August 21, 1996)     

Study of the cell cycle-dependent assembly of the DNA pre-replication centres in Xenopus egg extracts.

RPA is a cellular, three-subunit, single-stranded (ss) DNA binding protein, which assists T-antigen in the assembly of the pre-priming complex in the SV40 replication system. By immunodepletion and complementation, we have identified RPA as an essential factor for cellular DNA replication in Xenopus extracts. RPA assembles post-mitotically on the decondensing chromosomes into numerous subnuclear pre-replication centres (preRCs) which serve, upon formation of the nuclear membrane, as RCs for the initiation of DNA synthesis. By a variety of experiments including the use of isolated components, we demonstrate that an inactive cdc2-cyclin B kinase complex is essential to allow post-mitotic assembly of the preRCs. In contrast, the active cdk2-cyclin A kinase does not impede or facilitate the assembly of preRCs. Digestion analysis using the single-strand-specific P1 nuclease as well as competition experiments with ssDNA, reveal that replication-associated unwinding of the DNA, assisted by RPA, requires the formation of the nuclear membrane. The p21 cdk-interacting protein Cip1 appears to inhibit DNA replication prior to the unwinding DNA step, but after assembly of preRC and nuclear reconstruction.     

Mutant isolation of mouse DNA topoisomerase II alpha in yeast.

For characterizing in vivo functions of a mammalian protein, it is informative to obtain conditional mutations and apply them to the mouse genetic system. However, the isolation of conditional mutations has been quite difficult in cultured cells. We report here that functional expression of a heterologous mammalian gene in the yeast Saccharomyces cerevisiae provides a system for isolating mutated genes. We found that the cloned mouse TOP2 alpha cDNA, which encodes mouse DNA topoisomerase II (topo II) alpha, could rescue the lethal phenotype caused by yeast top2 null mutation. In order to generate and select temperature-sensitive mouse topo II alpha, an expression plasmid was mutagenized in vitro and was transformed, using the plasmid shuffling method, into the yeast strain, in which the endogenous TOP2 gene had been disrupted. We observed that one of such clone of yeast cells harboring a mutagenized mouse TOP2 alpha showed temperature-sensitive growth. Enzymatic assays and sequencing analysis revealed that this phenotype was caused by the thermosensitive nature of the mutant mouse protein, which has isoleucine at amino acid 961 instead of threonine. Therefore we have isolated the first conditional mutation in the mouse TOP2 alpha.     

Growth requirements of hyperthermophilic sulfur-dependent heterotrophic archaea isolated from a shallow submarine geothermal system with reference to their essential amino acids.

Three hyperthermophilic sulfur-dependent heterotrophs were isolated from a shallow submarine hydrothermal system at an inlet of Kodakara-jima island, Kagoshima, Japan. The isolates grew at 60 to 97 degrees C, with the optimum temperatures at 85 to 90 degrees C. Sensitivity to rifampin and the existence of ether lipids indicated that the isolates are hyperthermophilic archaea. Partial sequencing of the genes coding for 16S rRNA showed that the three isolates are closely related to the genus Thermococcus. They grew on proteinaceous mixtures, such as yeast extract, Casamino Acids, and purified proteins (e.g., casein and gelatin), but not on carbohydrates or organic acids as sole carbon and energy sources. Nine amino acids were essential for growth of isolate KS-1 (Thr, Leu, Ile, Val, Met, Phe, His, Tyr, and Arg). Isolate KS-2 required Lys in addition to the nine amino acids, and KS-8 required Lys instead of Tyr. In comparative studies, it was shown that Thermococcus celer DSM 2476 required 10 amino acids (Thr, Leu, Ile, Val, Met, Phe, Tyr, Trp, Lys, and Arg) while Pyrococcus furiosus DSM 3638 required only Ile and Val. The hyperthermophilic fermentative eubacterium Thermotoga neapolitana DSM 4359 did not require any amino acids for growth.     

Phylogenetic relationships among eutherian orders estimated from inferred sequences of mitochondrial proteins: Instability of a tree based on a single gene

The phylogenetic relationships among Primates (human), Artiodactyla (cow), Cetacea (whale), Carnivora (seal), and Rodentia (mouse and rat) were estimated from the inferred amino acid sequences of the mitochondrial genomes using Marsupialia (opossum), Aves (chicken), and Amphibia (Xenopus) as an outgroup. The overall evidence of the maximum likelihood analysis suggests that Rodentia is an outgroup to the other four eutherian orders and that Cetacea and Artiodactyla form a clade with Carnivora as a sister taxon irrespective of the assumed model for amino acid substitutions. Although there remains an uncertainty concerning the relation among Artiodactyla, Cetacea, and Carnivora, the existence of a clade formed by these three orders and the outgroup status of Rodentia to the other eutherian orders seems to be firmly established. However, analyses of individual genes do not necessarily conform to this conclusion, and some of the genes reject the putatively correct tree with nearly 5% significance. Although this discrepancy can be due to convergent or parallel evolution in the specific genes, it was pointed out that, even without a particular reason, such a discrepancy can occur in 5% of the cases if the branching among the orders in question occurred within a short period. Due to uncertainty about the assumed model underlying the phylogenetic inference, this can occur even more frequently. This demonstrates the importance of analyzing enough sequences to avoid the danger of concluding an erroneous tree.     

Phylogenetic place of guinea pigs: no support of the rodent-polyphyly hypothesis from maximum-likelihood analyses of multiple protein sequences

Graur et al.'s (1991) hypothesis that the guinea pig-like rodents have an evolutionary origin within mammals that is separate from that of other rodents (the rodent-polyphyly hypothesis) was reexamined by the maximum-likelihood method for protein phylogeny, as well as by the maximum-parsimony and neighbor-joining methods. The overall evidence does not support Graur et al.'s hypothesis, which radically contradicts the traditional view of rodent monophyly. This work demonstrates that we must be careful in choosing a proper method for phylogenetic inference and that an argument based on a small data set (with respect to the length of the sequence and especially the number of species) may be unstable.      

The giant panda is closer to a bear,judged by α- and β-hemoglobin sequences

Summary Using - and -hemoglobin sequences, we made a maximum likelihood inference as to the phylogenetic relationship among carnivores, including the two pandas, giant and lesser. Molecular phylogenetic studies up to 1985 had consistently indicated that the giant panda is more closely related to the bear than to the lesser panda. In 1986, however, a contradictory tree was constructed, using hemoglobins and so on, in which the two pandas link together (Tagle et al. 1986). In contrast to that tree, our conclusion supports the close relationship between bear and giant panda. The surface impression of a close relationship between the two pandas drawn from pairwise amino acid differences is explained by a rapid rate of hemoglobin evolution in the bear compared to that in the two pandas.Offprint requests to: T. Hashimoto     

A model for chromatin opening: stimulation of topoisomerase II and restriction enzyme cleavage of chromatin by distamycin.

Histone H1 preferentially and cooperatively binds scaffold-associated regions (SARs) in vitro via specific interactions with the numerous short A + T-rich tracts (A-tracts) contained in these sequences. Selective titration of A-tracts by the oligopeptide distamycin abolishes this interaction and results in a redistribution of H1. Similarly, treatment of intact cells and isolated nuclei with distamycin specifically enhances cleavage of internucleosomal linkers of SARs by topoisomerase II and restriction enzymes. The increased accessibility of these linkers is thought to result from the unfolding (or opening) of the chromatin fiber and to be due to a reduced occupancy by histone H1. Chromatin extraction and H1 assembly experiments support this view. We discuss a model whereby open, H1-depleted chromatin regions may be generated by titration of A-tracts by putative distamycin analogues; this local opening may spread to adjacent regions assuming highly cooperative H1-H1 interactions in chromatin.