Biomedical subjects

Miho Sugimori

Publications and source records attributed to Miho Sugimori.

5 recordsLinked to original sources

PubMed2005

Electronic microarray analysis of 16S rDNA amplicons for bacterial detection.

Electronic microarray technology is a potential alternative in bacterial detection and identification. However, conditions for bacterial detection by electronic microarray need optimization. Using the NanoChip electronic microarray, we investigated eight marine bacterial species. Based on the 16S rDNA sequences of these species, we constructed primers, reporter probes, and species-specific capture probes. We carried out two separate analyses for longer (533 bp) and shorter (350 and 200 bp) amplified products (amplicons). To detect simultaneously the hybridization signals for the 350- and 200-bp amplicons, we designed a common reporter probe from an overlapping sequence within both fragments. We developed methods to optimize detection of hybridization signals for processing the DNA chips. A matrix analysis was performed for different bacterial species and complementary capture probes on electronic microarrays. Results showed that, when using the longer amplicon, not all bacterial targets hybridized with the complementary capture probes, which was characterized by the presence of false-positive signals. However, with the shorter amplicons, all bacterial species were correctly and completely detected using the constructed complementary capture probes.

Bacteria↗

PubMed2005

Profiling and monitoring of microbial populations by denaturing high-performance liquid chromatography.

We describe a new molecular technique for the analysis of microbial species and complex microbial populations based on the separation of PCR-amplified 16S rDNA fragments by denaturing high-performance liquid chromatography (DHPLC). Using marine bacterial samples, we determined the optimum conditions for the analysis of bacterial species and the examination of complex bacterial assemblages obtained from different environments. The incorporation of a 40-bp GC clamp into the amplification primer was essential to effectively discriminate genetic differences in DHPLC-primers with a 20-, 10-, or 0-bp GC clamp length were less efficient. A 64.5 degrees C column temperature in DHPLC allowed optimal separation of species in a complex bacterial population. PCR-DHPLC analysis of bacterial assemblages demonstrated profiles with distinguishable peaks, which constituted the different populations and their degree of abundance. Fraction collection and DNA sequencing from profile peaks enabled bacterial identification. PCR-DHPLC analysis can also provide opportunities for describing bacterial communities, cloning bacteria, and monitoring bacterial populations in environments of interest.

Bacteria↗

PubMed2004

RERJ1, a jasmonic acid-responsive gene from rice, encodes a basic helix-loop-helix protein.

Differential screening of a cDNA library constructed using poly(A)(+) RNA from suspension-cultured rice cells treated with jasmonic acid (JA) for 1/2h yielded a cDNA of a gene tentatively named RERJ1 that is upregulated in response to exogenous JA. Northern blot analysis indicated that the RERJ1 mRNA levels peaked at 1/2-1h after the addition of jasmonic acid and then decreased gradually. RERJ1 encodes a transcriptional regulator with a basic helix-loop-helix motif. The phenotypes of transgenic rice plants overexpressing sense or antisense RERJ1 mRNA demonstrated that RERJ1 is involved in the growth inhibition of rice shoots caused by JA. Other biological functions of RERJ1 are discussed from an evolutionary standpoint.

Amino Acid Sequence↗

PubMed2002

Cloning and characterization of a jasmonic acid-responsive gene encoding 12-oxophytodienoic acid reductase in suspension-cultured rice cells.

In suspension-cultured rice ( Oryza sativaL.) cells, jasmonic acid (JA) functions as a signal transducer in elicitor N-acetylchitoheptaose-induced phytoalexin production. Differential screening of a cDNA library constructed using poly(A)(+) RNA from suspension-cultured rice cells treated with JA (10(-4) M) for 2 h yielded a cDNA for a gene that responded to exogenous JA by an increase in mRNA level. Nucleotide sequence analysis indicated that the cDNA encodes an homologue of the yeast Old Yellow Enzyme. The deduced amino acid sequence was very similar to the sequences of 12-oxophytodienoic acid reductases (OPR) 1 and 2 from Arabidopsis thaliana(AtOPR1 and AtOPR2) and OPR1 from tomato ( Lycopersicon esculentum) (LeOPR1). The cDNA-encoded protein purified from recombinant Escherichia coli cells as a hexahistidine-tagged fusion protein exhibited OPR activity similar to that of AtOPR1, AtOPR2, and LeOPR1, which catalyze reduction of (-)- cis-12-oxophytodienoic acid (OPDA) preferentially over (+)- cis-OPDA, a natural precursor of JA. Thus the rice enzyme was termed OsOPR1. The physiological roles of OsOPR1 are discussed. This is the first report of the cloning of an OPR gene from a monocot plant.

Cells, Cultured↗

PubMed2002

Cloning and characterization of cDNAs for the jasmonic acid-responsive Genes RRJ1 and RRJ2 in suspension-cultured rice cells.

Two cDNA clones for jasmonic acid (JA)-responsive genes, RRJ1 and RRJ2, were isolated by differential screening from suspension-cultured rice cells treated with JA for 2 h. The putative RRJ1 protein is completely identical to that of a putative rice cystathionine gamma-lyase, while the putative RRJ2 protein is highly similar in sequence to a rice pyruvate decarboxylase, PDC1.

Blotting, Northern↗