Koichi Yoshinaga

Competition between the Rad50 Complex and the Ku Heterodimer Reveals a Role for Exo1 in Processing Double-Strand Breaks but Not Telomeres

ABSTRACT The Mre11-Rad50-Nbs1(Xrs2) complex and the Ku70-Ku80 heterodimer are thought to compete with each other for binding to DNA ends. To investigate the mechanism underlying this competition, we analyzed both DNA damage sensitivity and telomere overhangs in Schizosaccharomyces pombe rad50-d, rad50-d pku70-d, rad50-d exo1-d, and pku70-d rad50-d exo1-d cells. We found that rad50 exo1 double mutants are more methyl methanesulfonate (MMS) sensitive than the respective single mutants. The MMS sensitivity of rad50-d cells was suppressed by concomitant deletion of pku70+ . However, the MMS sensitivity of the rad50 exo1 double mutant was not suppressed by the deletion of pku70+ . The G-rich overhang at telomere ends in taz1-d cells disappeared upon deletion of rad50+ , but the overhang reappeared following concomitant deletion of pku70+ . Our data suggest that the Rad50 complex can process DSB ends and telomere ends in the presence of the Ku heterodimer. However, the Ku heterodimer inhibits processing of DSB ends and telomere ends by alternative nucleases in the absence of the Rad50-Rad32 protein complex. While we have identified Exo1 as the alternative nuclease targeting DNA break sites, the identity of the nuclease acting on the telomere ends remains elusive.

Biological and chemical characteristics of the coral gastric cavity

All corals have a common structure: two tissue layers enclose a lumen, which forms the gastric cavity. Few studies have described the processes occurring inside the gastric cavity and its chemical and biological characteristics. Here, we show that the coral gastric cavity has distinct chemical characteristics with respect to dissolved O2, pH, alkalinity, and nutrients (vitamin B12, nitrate, nitrite, ammonium, and phosphate) and also harbors a distinct bacterial community. From these results, the gastric cavity can be described as a semi-closed sub-environment within the coral. Dissolved O2 shows very low constant concentrations in the deepest parts of the cavity, creating a compartmentalized, anoxic environment. The pH is lower in the cavity than in the surrounding water and, like alkalinity, shows day/night variations different from those of the surrounding water. Nutrient concentrations in the cavity are greater than the concentrations found in reef waters, especially for phosphate and vitamin B12. The source of these nutrients may be internal production by symbiotic bacteria and/or the remineralization of organic matter ingested or produced by the corals. The importance of the bacteria inhabiting the gastric cavity is supported by the finding of a high bacterial abundance and a specific bacterial community with affiliation to bacteria found in other corals and in the guts of other organisms. The findings presented here open a new area of research that may help us to understand the processes that maintain coral health.

Chlorella chloroplast DNA sequence containing a gene for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase and a part of a possible gene for the β' subunit of RNA polymerase.

The sequence of a 2782 bp fragment of the chloroplast genome of Chlorella ellipsoidea has been determined. The region includes the entire gene (rbcL) for the large subunit (LS) of ribulose-1,5-bisphosphate carboxylase/oxygenase and a sequence (rpoC-like) similar to part of the gene for the subunit of E. coli RNA polymerase which is oriented in same direction as rbcL. The arrangement is rpoC-like — 446 bp — rbcL. The rbcL gene codes for a polypeptide of 475 amino acids whose sequence shows 88% homology with those of tobacco and spinach, 94% homology with that of Chlamydomonas, and 85% homology with that of Anacystis. The putative rbcL promoter sequence has homology with E. coli promoter sequences and its putative terminator sequence is capable of forming a stem-and-loop structure.

Intraspecific Sequence Variation of Chloroplast DNA Reflecting Variety and Geographical Distribution of Polygonum cuspidatum (Polygonaceae) in Japan

Polygonum cuspidatum in Japan, we analyzed the chloroplast DNA sequences of a region from the rbcL to the accD gene (ca. 1,420 bp), and found nucleotide variations at 22 sites in 68 samples. The phylogenetic relationship deduced from the sequence variations revealed the existence of at least five groups. The first group consisted of P. cuspidatum var. cuspidatum in the central part of Honshu; in Nagano, Yamanashi, and Shizuoka. The second, a sister of the first, consisted of those plants in Shizuoka-Itoigawa Line. The third group consisted of plants in the northern part of Japan including P. sachalinense in Hokkaido, P. cuspidatum var. cuspidatum in Aomori and var. uzensis in Akita. The fourth consisted of var. uzensis in the Tohoku District. The fifth consisted of var. terminalis in the Izu Islands. P. cuspidatum are differentiated according to their distribution, and two varieties, var. terminalis and var. uzensis, are differentiated genetically. Polygonum sachalinensis, a distinct species morphologically, fell into the accessions of P. cuspidatum on the phylogenetic tree obtained in the present study.

Double-strand scission of DNA involved in thymineless death of Escherichia coli 15 TAU.

Abstract Double-strand DNA scissions occurring during thymineless death of Escherichia coli 15 TAU were assayed by neutral sucrose gradient sedimentation of [ 3 H]thymidine-labelled DNA isolated from cells lysed under mild conditions. It was found that, under thymineless conditions, the sedimentation rate of DNA started to decrease only after the cells began to lose their viability. When thymine was added to thymine-starved cultures, the progress of thymineless death was stopped and viable cells began to increase. However, the sedimentation rate of the labelled DNA was not restored to that of the original DNA. These results indicate (1) that thymineless death is accompanied by double-strand scission of DNA, (2) that thymine starvation does not per se cause the DNA scissions but only when cell death ensues, and (3) that the broken DNA cannot be converted back into its original form. It is suggested that double-strand scission of DNA is the principal cause for thymineless death in this case.

Purification and properties of trehalose-synthesizing enzyme from Pseudomonas sp. F1

The trehalose-synthesizing enzyme, which catalyzes the conversion of maltose to trehalose by intramolecular transglucosylation, was purified from a bacterium, Pseudomonas sp. F1. Its molecular mass was estimated to be 250 kDa by gel filtration and 67 kDa by SDS-polyacrylamide gel electrophoresis, and its pI was 5.8. The native enzyme may consist of 4 subunits. The enzyme was active on maltose and trehalose among saccharides tested as substrates. The N-terminal amino acid of the enzyme was threonine.

Nucleotide sequence ofatpB,rbcL,trnR,dedB andpsaI chloroplast genes from a fernAngiopteris lygodiifolia: a possible emergence of Spermatophyta lineage before the separation of Bryophyta and Pteridophyta

To elucidate the evolutionary relationship between the Spermatophyta, Pteridophyta and Bryophyta, we cloned a fragment of chloroplast DNA from the fernAngiopteris lygodiifolia (Pteridophyta) and determined its nucleotide sequence. The fragment contained theatpB,rbcL,trnR-CCG,dedB andpsaI genes. Comparisons of the deduced amino acid and nucleotide sequences of these genes from the three plant groups indicate thatAngiopteris sequences are more closely related to those of Bryophyta species (85% identity on average) than to those of seed plants (76% identity on average), supporting a hypothesis that the Bryophyta and Pteridophyta diverged more recently from one another than their common progenitor diverged from that of the Spermatophyta.

A temperature-sensitive mutant of Escherichia coli with an altered RNA polymerase β′ subunit

Abstract A temperature-sensitive RNA polymerase mutant of Escherichia coli K-12 was isolated. Subunit reconstitution experiments showed that the mutation was located in the gene for β′ subunit. RNA polymerase purified from the mutant was stable and showed reversible temperature sensitivity in vitro . At high temperature, the initiation of RNA synthesis was impaired and the degree of inhibition was different with templates used. These results suggested that the β′ subunit had an essential role for promoter selection.

Organization of chloroplast ribosomal RNA genes and in vitro self-splicing activity of the large subunit rRNA intron from the green alga Chlorella vulgaris C-27

Abstract Sequencing of the rRNA gene (rrn) cluster of Chlorella vulgaris C-27 chloroplasts has revealed a striking organizational difference in comparison to another species of the same genus, Chlorella ellipsoidea C-87. The rrn23 gene in C. vulgaris is also split. However, the 815-bp intervening sequence in this gene has been identified as a group-I intron. An in vitro rrn23 transcript containing the entire intron and parts of flanking exon sequences is able to self-splice in vitro in the presence of GTP and Mg++. Accurate ligation of the exons has been confirmed by sequencing the cDNA of the spliced products. GTP labelling of total Chlorella RNA in vitro has revealed that the number of self-splicing RNAs present in Chlorella chloroplasts is limited compared to that found in other green algal species.

Kinetic analysis of the effect of (-)-epigallocatechin gallate on the DNA scission induced by Fe(II).

The DNA strand scission induced by Fe(II) in a citrate buffer solution and the effect of (−)-epigallocatechin gallate (EGCg) were kinetically analyzed. The rate of consumption of dissolved oxygen by Fe(II) in each of these solutions was measured and paralleled that DNA scission. Coordinated EGCg accelerated these reactions. Curves of the time-course characteristics of DNA scission were simulated by using the rate constant of oxygen consumption and by assuming that scission with the hydroxyl radical (OH), which was formed from the dissolved oxygen, proceeded competitively with the scavenging of OH by citrate, Cl− ions and EGCg added. Free EGCg acted as a DNA scission inhibitor to scavenge OH, in contrast to the case of the coordinated one. This analysis is useful for estimating the rate constant of the reaction between an antioxidant and OH, and might provide a new method for measuring the OH-scavenging activity.