Shigeyuki Ichihara

A Rice Semi-Dwarf Gene, Tan-Ginbozu (D35), Encodes the Gibberellin Biosynthesis Enzyme, ent-Kaurene Oxidase

A rice (Oryza sativa L.) semi-dwarf cultivar, Tan-Ginbozu (d35Tan-Ginbozu), contributed to the increase in crop productivity in Japan in the 1950s. Previous studies suggested that the semi-dwarf stature of d35Tan-Ginbozu is caused by a defective early step of gibberellin biosynthesis, which is catalyzed by ent-kaurene oxidase (KO). To study the molecular characteristics of d35Tan-Ginbozu, we isolated 5 KO-like(KOL) genes from the rice genome, which encoded proteins highly homologous to Arabidopsis and pumpkin KOs. The genes (OsKOL1to5) were arranged as tandem repeats in the same direction within a 120 kb sequence. Expression analysis revealed that OsKOL2 and OsKOL4 were actively transcribed in various organs, while OsKOL1 and OsKOL5 were expressed only at low levels; OsKOL3 may be a pseudogene. Sequence analysis and complementation experiments demonstrated that OsKOL2corresponds to D35. Homozygote with null alleles of D35showed a severe dwarf phenotype; therefore, d35Tan-Ginbozu is a weak allele of D35. Introduction of OsKOL4 into d35Tan-Ginbozu did not rescue its dwarf phenotype, indicating that OsKOL4 is not involved in GA biosynthesis. OsKOL4 and OsKOL5 are likely to take part in phytoalexin biosynthesis, because their expression was promoted by UV irradiation and/or elicitor treatment. Comparing d35Tan-Ginbozu with other high yielding cultivars, we discuss strategies to produce culm architectures suitable for high crop yield by decreasing GA levels.

Reduction of protein degradation by use of protease-deficient mutants in cell-free protein synthesis system of Escherichia coli.

In an Escherichia coli in vitro transcription/translation system, the degradation of produced proteins is often caused by endogenous proteases from E. coli extracts. To reduce the extent of this degradation, several extracts were prepared from E. coli mutants that genetically lacked DegP, OmpT, or Lon proteases. Then, these extracts were used with 14C-leucine in a system for synthesizing single-chain Fv against gp120 (anti-gp120), and phospholipase D (PLD) of Streptomyces antibioticus. The proteins synthesized in vitro were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and autoradiography. The use of extracts from mutants that were deficient in the structural genes encoding OmpT and Lon markedly repressed the degradation of anti-gp120. Similarly, extracts from degP- and ompT-deleted mutants were able to significantly stabilize in vitro-synthesized PLD, which otherwise disappeared within 30 min. Such protease-deficient mutants were suggested to be useful for preventing the degradation of heterologous proteins in in vitro systems.

Isolation of rat mitochondrial transcription factor A (r-Tfam) cDNA

We have isolated rat mitochondrial transcription factor A (Tfam; formerly known as mtTFA) cDNA clones from a rat cerebellum cDNA library using human Tfam cDNA as a probe. The deduced amino acid sequence of r-Tfam shows 62% and 89% overall identity to human and mouse Tfam, respectively. We also show the presence of two r-Tfam isoforms in testis as for mouse. Our findings suggest that the mechanisms underlying transcription of mitochondrial genes are conserved among rat, mouse, and human.

Effects of various culture environments on expression of major outer membrane proteins from Porphyromonas gingivalis

We examined the effects of various culture environments on major outer membrane proteins from Porphyromonas gingivalis ATCC 33277. Major outer membrane protein patterns on gel electrophoresis showed little difference over the culturable range of osmolarity and pH. With elevated temperature or prolonged culture, the intensities of the gingipain bands decreased; however, bands of RagA, RagB and the putative porins were relatively stable. Similar results were observed with several different culture media. Although the precise functions of RagA, RagB and the putative porins are unknown, these factors may be strongly related to the initiation and progression of adult periodontitis.

alpha-, beta- or gamma-chain-specific RNA interference of laminin assembly in Drosophila Kc167 cells.

Drosophila laminin alphabetagamma trimer assembly in Kc167 cells was perturbed by chain-specific RNA interference (RNAi). The intracellular pool of alpha and gamma chains remained unchanged under beta-chain RNAi by lipofection of double-stranded RNA encoding a beta-chain partial sequence. This was also the case for the intracellular pool of alpha and beta chains under gamma-chain-specific RNAi. Nonetheless, the intracellular pool of beta and gamma chains increased markedly under alpha-chain-specific RNAi. Non-reducing SDS/PAGE revealed that some of the increased beta and gamma chains migrated as disulphide-linked betagamma dimers but that the rest migrated as monomers. Since the monomeric beta and gamma bands detected under alpha-chain RNAi were denser than the beta band under gamma-chain RNAi and the gamma band under beta-chain RNAi, respectively, beta and gamma also appeared to accumulate by forming betagamma dimers without the disulphide linkage. We suggest that interconversion of these betagamma dimers is crucial for the replaceable and selective assembly of the alpha chain for alphabetagamma trimer formation.

Syndecan‐dependent binding of Drosophila hemocytes to laminin α3/5 chain LG4‐5 modules: potential role in sessile hemocyte islets formation

Heparin‐column chromatography and elastase‐digestion of medium from hemocyte Kc167 gave Drosophila laminin α3/5βγ trimer, α3/5LG2‐3 and α3/5LG4‐5 modules with eluting NaCl concentrations of 450, 280 and 450 mM, respectively. Kc167 cells bound dish surface with α3/5βγ trimer or α3/5LG4‐5, but not with α3/5LG2‐3 modules. Cell binding was counteracted by treating with heparin or heparan sulfate. RNA interference of syndecan in Kc167 cells impaired the binding, but that of dally or dally‐like did not. Green fluorescent protein‐expressing hemocytes also bound surface with α3/5βγ trimer or α3/5LG4‐5 module. Thus, syndecan‐dependent binding of hemocytes to laminin may have a potential role in sessile hemocytes islets formation in T2‐A8 segments of Drosophila.

The Evolution of the Phenylacetic Acid Degradation Pathway in Bacteria

The phenylacetic acid (PhAc) degradation pathway becomes an interesting model for the catabolism of aromatic compounds. To determine the molecular basis for this environmentally important process, we did a phylogenic analysis based on the PhAc CoA ligase gene. It suggests that the PhAc CoA ligase genes are distributing widely and subject to frequent lateral gene transfer within and across bacterial phylum.

Apoptotic Cell Death in the Fission Yeast Schizosaccharomyces pombe Induced by Valproic Acid and Its Extreme Susceptibility to pH Change

Schizosaccharomyces pombe treated with valproic acid died with apoptotic markers such as DNA fragmentation, loss of a mitochondrial electrochemial gradient and chromatin condensation, independently of metacaspase, a yeast homolog of metazoan caspase. Sensitivity to valproic acid was strongly dependent on growth phase. Cells in a later growth phase were much more sensitive to valproic acid than those in an earlier one. Altering the pH of the medium with HCl and with NaOH also caused remarkable changes in sensitivity. Cells in an acidic medium were more sensitive to valproic acid. This pH-dependent change in sensitivity did not require de novo protein synthesis, and a change in pH 60 min after the administration of valproic acid affected sensitivity. These results suggest that the intracellular cell death process was susceptible to extracellular pH. Although a sir2 mutant of Saccharomyces cerevisiae has been reported to be resistant to valproic acid, mutations in sir2 did not affect the sensitivity to valproic acid of S. pombe.

Sterilization of liquid by high electric field pulse

There are two methods for sterilization of foods, heating and non-heating. The heating sterilization is now widely used for liquid food such as milk and juice. However this method has some problems. One of them is the changes in nutrition and quality of food caused by heating up to temperature for example 63°C to kill bacteria. The large energy consumption is also a drawback of heating sterilization; the heating sterilization needs to heat up whole volume of liquid. The non-heating sterilization such as UV irradiation, ion beam irradiation, and chemical methods are proposed to avoid these problems of heating method. The pulse sterilization, one of the non-heating methods, attracts attentions because it causes little change in nutrition of liquid food, and low energy consumption. We have studied the sterilization of liquid using high electric field pulse. Sample was liquid medium including Escherichia coli. E. coli was used because of easy cultivation. We used nonpoisonous K coli. We applied high voltage pulse to a coaxial cylindrical tank containing sample liquid. After application of 50 pulses, the survival rate of K coli was reduced to 0.01%. This value of 0.01% is far superior to 1%, which is reported for milk sterilized at 63°C for 30 minutes.

Electric field pulse sterilization of liquid food

We have studied the sterilization of liquid using high electric field pulses. The sample was liquid medium including Escherichia coli. We applied high voltage pulses to a coaxial cylindrical tank containing the sample liquid. After the application of 50 pulses, the survival rate of E. coli was reduced to 3.9×10 −3%. This value of 3.9×10 −3% is far superior to 1%, which is provided for milk sterilized at 63°C for 30 minutes. The length of E. coli cell, in most cases, becomes longer after the pulses application. It seems that the high electric field pulse application prevents E. coli from cell division.