Hideki Yamamoto

Identification of protein kinase A catalytic subunit β as a novel binding partner of p73 and regulation of p73 function

Post-translational modifications play a crucial role in regulation of the protein stability and pro-apoptotic function of p53 as well as its close relative p73. Using a yeast two-hybrid screening based on the Sos recruitment system, we identified protein kinase A catalytic subunit β (PKA-Cβ) as a novel binding partner of p73. Co-immunoprecipitation and glutathione S-transferase pull-down assays revealed that p73α associated with PKA-Cβ in mammalian cells and that their interaction was mediated by both the N- and C-terminal regions of p73α. In contrast, p53 failed to bind to PKA-Cβ. In vitro phosphorylation assay demonstrated that glutathione S-transferase-p73α-(1–130), which has one putative PKA phosphorylation site, was phosphorylated by PKA. Enforced expression of PKA-Cβ resulted in significant inhibition of the transactivation function and pro-apoptotic activity of p73α, whereas a kinase-deficient mutant of PKA-Cβ had no detectable effect. Consistent with this notion, treatment with H-89 (an ATP analog that functions as a PKA inhibitor) reversed the dibutyryl cAMP-mediated inhibition of p73α. Of particular interest, PKA-Cβ facilitated the intramolecular interaction of p73α, thereby masking the N-terminal transactivation domain with the C-terminal inhibitory domain. Thus, our findings indicate a PKA-Cβ-mediated inhibitory mechanism of p73 function.

Rapid determination of the nucleotide sequences of potyviral coat protein genes using semi-nested RT-PCR with universal primers

A rapid method (<20xa0h) for determining the nucleotide sequences of potyviral coat protein (CP) genes was developed. The procedure involves reverse transcription using total RNA from a potyvirus-infected plant, followed by polymerase chain reaction (PCR) with the universal primers Sprimer/M4. The expected fragment, purified from an agarose gel, is used in semi-nested PCR with three degenerate primer sets: Sprimer/D1000, Sprimer/RYAFDFY-C and U341/M4. The three purified fragments are directly sequenced using the respective primers D1000, RYAFDFY-C and U341, then the complete CP sequence is assembled. All 11 potyviruses tested were efficiently sequenced by this method.

A Missense Mutation in Tomato mosaic virus L11A-Fukushima Genome Determines Its Symptomless Systemic Infection of Tomato

The genome of an attenuated isolate L11A-Fukushima (L11A-F) of Tomato mosaic virus was cloned, its complete nucleotide sequence was determined, and full-length clone of L11A-F was then assembled. Its transcripts systemically infected tomato without causing any symptom development. When a missense back mutation at the nucleotide correspondent to nucleotide 2350 of the genome was introduced into the clone, its transcripts produced distinct mosaic on the upper leaves of the inoculated tomato, and virus accumulation in the upper leaves increased about five times. The missense mutation in the L11A-F genome was thus confirmed to be sufficient to attenuate its virulence on tomato.

Characterization of the nuclear DNA of Phialophora gregata ff.sp, adzukicola and sojae

Phialophora gregata nuclear (n) DNA was characterized by physical methods. The nDNA of f.sp.adzukicola was shown to be larger than that of f.sp.sojae, 2.9 and 2.1 × 1010 Da, respectively. The amounts of repetitive sequence and AT-rich region in the nDNA were also larger in f.sp.adzukicola than f.sp.sojae. These results indicate that the nuclear genome organization of the two formae speciales is differentiated.

Characterization of a novel potyvirus tentatively named Ornithogalum virus 3, successfully isolated from O. thyrsoides co-infected with two other potyviruses by single-aphid inoculation

A potyvirus tentatively named Ornithogalum virus 3 (OV-3) was successfully isolated by single-aphid transmissions from O. thyrsoides mix-infected with OV-3, Ornithogalummosaicvirus (OrMV) and Ornithogalum stripe mosaic virus (OrSMV). OV-3, a flexuous, rod-shaped particle of ca. 690xa0nm, was sap and aphid transmissible. The virus had a narrow host range and caused necrotic mosaic on O. thyrsoides under cold conditions. We therefore propose the name Ornithogalum necrotic mosaic virus (OrNMV) for OV-3. A synergistic increase in symptom severity was apparent on O. thyrsoides mix-infected with OrSMV/OrNMV, but not with either OrMV/OrNMV or OrMV/OrSMV.

Discrimination between Virulent and Attenuated Isolates of Tomato mosaic virus by Restriction Fragment Length Polymorphism

The three missense mutations on the gene for the 130-K protein of Tomato mosaic virus (ToMV) L11A have been thought to be responsible for the attenuation of its virulence. The Eco47I RFLP detecting the missense mutation at 2349 successfully discriminated L11A and its derivative attenuated isolates from ToMV virulent ones. RFLP analysis and mismatch amplification assay detecting the missense mutations at 1117 and 2754, respectively, could not discriminate some of the attenuated isolates from the virulent ones. These results indicated that, of the three missense mutations, only the one at 2349 was conserved in all the L11A-derivative attenuated isolates.

Isolation and characterization of a novel potyvirus tentatively named Ornithogalum virus 2

A novel potyvirus, tentatively named Ornithogalum virus 2 (OV-2) because only its nucleotide sequence of the coat protein gene has been revealed, was isolated for the first time from Ornithogalum thyrsoides. OV-2 had a flexuous particle (700–740xa0nm in length) and was sap and aphid transmissible. The virus had a narrow host range; of 36 test plants in 12 families, only O. thyrsoides and O. dubium were infected. Because the virus caused characteristic stripe mosaic on O. thyrsoides, we propose Ornithogalum stripe mosaic virus (OrSMV), instead of OV-2 for the proper name of the virus.

Differential susceptibility of Phialophora gregata ff. sp. adzukicola and sojae to antimicrobial chemicals

The susceptibility ofPhialophora gregata ff.sp.adzukicola andsojae to antimicrobial chemicals was investigated. The minimum inhibitory concentrations (MICs) of benomyl, chloramphenicol, CuSO4, cycloheximide and perchlorate for mycelial growth were the same for the two formae speciales. The MIC of hygromycin against f.sp.adzukicola was slightly lower than that against f.sp.sojae, and the latter was more resistant to iprodion than the former. Susceptibility to nystatin was markedly different: ff.sp.adzukicola andsojae had relative growth values of 3–20% and 59–93% at 100 µg/ml, respectively, and this difference could be used to differentiate the two formae speciales.

Characteristics for Practical Use of Attenuated Isolate UA-Fukushima of Tomato mosaic virus

L11A-Fukushima (L11A-F) derived from attenuated isolate LuA of Tomato mosaic virus (ToMV) has the highest ability to cross protect against virulent ToMV among LuA and its derivatives and is stably inherited. Growth, yield, fruit quality and symptom attenuation of inoculated tomato plants did not differ significantly between L11A-F and L11A. The infectivity of progeny viruses in tomato infected with LuA-F was less than 4% of that with virulent ToMV. From these results, L11A-F appears to possess the properties necessary for practical use. To manage L11A-F strictly, a PCR-based assay to detect trace contamination of virulent ToMV in L11A-F preparations was established.