<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>-mediated transformation of the violet root-rot fungus, <Emphasis Type="Italic">Helicobasidium mompa</Emphasis>, and the effect of activated carbon

Agrobacterium tumefaciens-mediated transformation (ATMT) has been successfully applied to the violet root-rot fungus Helicobasidium mompa, which is the causal agent of violet root-rot disease. The A. tumefaciens strains carried a binary plasmid vector containing the hygromycin B phosphotransferase gene (hph) controlled by the heterologous fungal Agaricus bisporus P-gpd (glyceraldehyde-3-phosphate dehydrogenase) promoter and the trpC terminator. The transformation system was optimized using defined cocultivation conditions. When H. mompa strain V17 was cocultivated with A. tumefaciens strain AGL-1 using 5% agar, we obtained more hygromycin-resistant colonies than with strains EHA105 or MAFF301222 using 2% agar. In addition, our results suggest that the activated carbon is necessary in ATMT to reduce background growth of H. mompa. The presence of the hph gene in transformants was detected by polymerase chain reaction (PCR), and single-copy integration of the marker gene was demonstrated by Southern blot analysis. Thus, the ATMT system can be considered a promising tool for insertional mutagenesis studies of H. mompa.     

Agrobacterium tumefaciens-mediated transformation of embryogenic tissue and transgenic plant regeneration in Chamaecyparis obtusa Sieb. et Zucc.

A genetic transformation procedure for Chamaecyparis obtusa was developed after co-cultivation of embryogenic tissues with disarmed Agrobacterium tumefaciens strain C58/pMP90, which harbours the sgfp (synthetic green fluorescent protein) visual reporter and nptII (neomycin phoshotransferase II) selectable marker genes. The highest transformation frequency was 22.5 independent transformed lines per dish (250 mg embryogenic tissue) following selection on kanamycin medium. Transgenic plantlets were regenerated through the maturation and germination of somatic embryos. The intensity of GFP fluorescence, observed under a fluorescence microscope, varied from very faint to relatively strong, depending on the transgenic line or part of the transgenic plant. The integration of the genes into the genome of regenerated plantlets was confirmed by Southern blot analysis.     

Genetic variations in Lycoris radiata var. radiata in Japan

The genetic variations of Lycoris radiata var. radiata, a completely sterile triploid from Japan, were examined by comparing the nucleotide sequences of genomic DNA regions in 11 triploid strains sampled from Japan and four triploid strains sampled from China, and in two diploid strains of Lycoris radiata var. pumila, which is endemic to China and fertile. For this purpose, two genes were analyzed, the lectin gene in the nuclear genome and the maturase gene in the chloroplast genome. A clear genetic constancy was observed in their DNA nucleotide sequences. For both genes, completely identical nucleotide sequences were detected in the 11 Japanese and four Chinese triploid strains and also between the two Chinese diploid strains. However, some genetic variations were observed between the Japanese and Chinese triploid strains, and between the triploid and diploid strains. These results are consistent with the findings obtained from previous chromosome karyotype analyses and allozyme analyses. In addition, in our preliminary FISH analysis of the physical mapping of the rRNA gene family, the 18S-5.8S-26S rRNA and 5S rRNA loci were localized on six and four chromosomes, respectively. Regarding the 18S-5.8S-26S rRNA loci, two were associated with two SAT chromosomes. The remaining four were distinguished by having no secondary constriction. Localization of 5S rRNA loci to chromosome spreads revealed three sites on the proximal part of the long arm of three acrocentric chromosomes and one site on the distal part of the long arm of the SAT chromosome; the latter site was juxtaposed to the 18S-5.8S-26S rRNA loci. These findings indicate that L. radiata var. radiata is not a typical autotriploid. The present paper discusses the possible origin of L. radiata var. radiata from a diploid variety of L. radiata var. pumila, based on the molecular cytogenetic analysis and DNA sequence analysis.     

Al(3+) interaction sites of calmodulin and the Al(3+) effect on target binding of calmodulin

The interaction between calmodulin (CaM) and Al(3+) was studied by spectroscopic methods. Heteronuclear two-dimensional NMR data indicated that peaks related to the both lobes and middle of the central helix of CaM are largely affected by Al(3+). But chemical shift perturbation suggested that overall conformation of Ca(2+)-loaded CaM is not changed by Al(3+) binding. It is thought that Al(3+) interaction to the middle of the central helix is a key for the property of CaM's target recognition. If the structure and/or flexibility of the central helix are/is changed by Al(3+), target affinity to CaM must be influenced by Al(3+). Thus, we performed surface plasmon resonance experiments to observe the effect of Al(3+) on the target recognition by CaM. The data clearly indicated that target affinity to CaM is reduced by addition of Al(3+). All the results presented here support a hypothesis that Al(3+) may affect on the Ca(2+) signaling pathway in cells.     

The specific p38 mitogen-activated protein kinase pathway inhibitor FR167653 keeps insulitis benign in nonobese diabetic mice

The p38 mitogen-activated protein kinase (MAPK) pathway is important in Th1 immunity, macrophage activation, and apoptosis. Since they may be associated with beta-cell destruction during the development of type 1 diabetes, we investigated the role of the p38 MAPK pathway in female nonobese diabetic (NOD) mice. Phosphorylated p38 MAPK was observed immunohistochemically in CD4+ cells that had infiltrated into the islets and part of beta-cells, increasing in proportion to the severity of insulitis. Continuous oral administration of 0.08% FR167653, a specific p38 MAPK pathway inhibitor, significantly reduced the ex vivo production of interferon-gamma by splenic Th1 cells without affecting interleukin-4 production by Th2 cells. FR167653 administration from 4-30 weeks of age prevented NOD mice from developing diabetes without affecting the severity of insulitis. Treatment with FR167653 after insulitis had developed (i.e. from 10-30 weeks of age) also prevented diabetes, further suggesting that treatment with the p38 MAPK pathway inhibitor keeps insulitis benign in NOD mice, partly by inhibiting Th1 immunity. These findings suggest that p38 MAPK is a key mediator that switches insulitis from benign to destructive in the development of type 1 diabetes.     

p51/p63, a novel p53 homologue, potentiates p53 activity and is a human cancer gene therapy candidate

BACKGROUND: p51 (p73L/p63/p40/KET), a recently isolated novel p53 homologue, binds to p53-responsive elements to upregulate some p53 target genes and has been suggested to share partially overlapping functions with p53. p51 may be a promising candidate target molecule for anti-cancer therapy. METHODS: In this study, we adenovirally transduced p51A cDNA into human lung, gastric and pancreatic cancer cells and analyzed the intracellular function of p51 in anti-oncogenesis in vitro and in vivo. RESULTS: Overexpression of p51A revealed an anti-proliferative effect in vitro in all the cancer cells examined in this study. The anchorage-dependent and -independent cell growth of EBC1 cells carrying mutations in both p51 and p53 was suppressed and significant apoptosis following adenoviral transduction with p51 and/or p53 was seen. This growth suppression was cooperatively enhanced by the combined infection with adenoviral vectors encoding both p51 and p53. Furthermore, p51 activated several, but not all, p53-inducible genes, indicating that the mechanisms controlling p51- and p53-mediated tumor suppression differed. CONCLUSIONS: Our observations indicate that, although p51 exhibited reduced anti-oncogenetic effects compared with p53, it cooperatively enhanced the anti-tumor effects of p53. Our results suggest that p51 functions as a tumor suppressor in human cancer cells in vitro and in vivo and may be useful as a potential tool for cancer gene therapy.     

Genetic variations in the chloroplast genome and phylogenetic clustering of Lycoris species

The genus Lycoris of Amaryllidaceae comprises approximately 20 species that are distributed only in the moist warm temperate woodlands of eastern Asia. The objectives of this study were: (1) to clarify the phylogeny of the Lycoris species by using the definitive DNA sequencing method and (2) to examine the possible maternal donor of the hybrid origin Lycoris species and the Japanese triploid strains of Lycoris radiata var. radiata. The nucleotide sequence of the maturase K (matK) gene and the noncoding intergenic spacer (IGS) between the atpB and rbcL genes in the chloroplast genome were determined in a total of 27 strains of 11 species of the genus Lycoris. Variation among taxa was mainly due to nucleotide substitution, although deletions and an insertion were found in the IGS. For two chloroplast regions, the phylogenetic trees showed essentially similar topology, indicating the existence of four clades, I, II, III, and IV. For all the species except L. radiata, intraspecific variation was smaller than interspecific variation. For L. radiata, triploid strains were divided into clades I and II, and diploid strains were divided into clades I and IV. This implies that the diploid species of L. radiata var. pumila is a probable ancestral species. The clustering indicated that the chloroplast genome has not evolved in parallel with the karyotype in genus Lycoris. Regarding the hybrid origin species, the maternal parents of L. squamigara, L. albiflora and L. rosea were revealed to be L. longituba, L. radiata and L. radiata var pumila, respectively. We also suggest that a diploid strain of L. radiata var. pumila in clade I might be a candidate of the maternal donor of the Japanese triploid strains. A possible model of the maternal donor of Lycoris species is proposed.     

Molecular cloning of cDNA encoding APC11, a catalytic component of anaphase-promoting-complex (APC/C), from goldfish (Carassius auratus), and establishment of in vitro ubiquitinating system

Destruction of cyclin B is required for exit from mitosis and meiosis. A cyclin-degrading system, including anaphase-promoting-complex/cyclosome (APC/C), has been shown to be responsible for cyclin B destruction. Here we present the cloning, sequencing, and expression analysis of goldfish (Carassius auratus) APC11, which encodes the catalytic component of APC/C from goldfish ovary. The cloned cDNA is 348 bp long and encodes 88 amino acids. The deduced amino acid sequence is highly homologous to APC11 from other species. The expression of mRNA for APC11 was ubiquitous among tissues, as opposed to that of mRNA for E2-C, which occurred at a very high level in the ovary. Recombinant goldfish APC11 possesses ubiquitinating activity against cyclin B. We established an in vitro ubiquitinating system of proteins composed of purified recombinant E1, E2-C, and APC11 from goldfish. The reconstructed system for these ubiquitinating enzymes makes it feasible to elucidate the molecular mechanism of cyclin B degradation.