Satoshi Futo

Possible participation of pICln in the regulation of angiogenesis through alternative splicing of vascular endothelial growth factor receptor mRNAs

In this study, the authors applied a modified Antisense Display method to human vascular endothelial cells (ECs) in culture to isolate new angiostatic genes. Screening of a 10mer antisense oligodeoxyribonucleotide (oligo) repertoire identified a subpool that consistently stimulated EC growth. Subsequent screening of oligos with increasing chain length led to the isolation of a unique growth-stimulatory 14mer, 5-TTCCACATCATATT-3. cDNA/EST data-base search and expression analyses in ECs indicated pICln as the corresponding gene. A longer unique antisense oligo against a different region of pICln mRNA was found to also enhance EC growth and tube formation and to decrease mRNAs for soluble Flt-1 and neuropilin-1 vascular endothelial growth factor (VEGF) receptors, the angiostatic factors that are generated by alternative RNA splicing. Conversely,pICln overexpression suppressed EC growth and increased the mRNAs for both soluble Flt-1 and soluble neuropilin-1. The present findings thus suggest that pICln plays a role in autocrine regulation of angiogenesis, probably through alternative splicing.

Identification of Single Base-pair Mutation on uidA Gene of Escherichia coli O157:H7 by Peptide Nucleic Acids (PNA) Mediated PCR Clamping

Peptide Nucleic Acids (PNA) is a new type of DNA analogue with a peptide backbone. We developed a rapid identification system of Escherichia. coli O157:H7 using PNA mediated PCR clamping. Firstly, we confirmed a single nucleotide alteration in the uidA gene (T93G), which is specific to E. coli O157: H7. We designed forward mutant DNA primer, wild type PNA, and a reverse DNA primer corresponding to the uidA sequence. PCR cycle consisted of four steps including dual annealing temperatures, 57°C and 45°C. Among 20 E. coli strains with various serotypes and 4 neighboring strains, the amplified bands (517 bp) were detected only in E. coli O157:H7 strains. PNA has specifically inhibited the PCR amplification from a wild type uidA gene. We successfully developed a multiplex PCR system, which detects both shigatoxin (stx) and uidA genes at once, to get reliable results by easier and rapid operation. We also analyzed kinetic parameters of PNA/DNA association using surface plasmon resonance and melting temperature using fluorescence resonance energy transfer (FRET). We discussed a selection mechanism of PCR clamping from these results.

Rapid selection of nonhotspot mutants among hisD+ revertants of Salmonella typhimurium TA98 in Ames test by peptide nucleic acid (PNA)-mediated PCR clamping.

Ames test is the most popular method of assessing mutagenicity using Salmonella typhimurium as an indicator. Recently, sequence analyses have been introduced for the investigation of mutation mechanisms. Most revertants (>70%) carry 2-bp deletion within an 8-bp CG repeat in hisD (hotspot mutation) in the Ames test using S. typhimurium TA98. We developed a new specific amplification method for nonhotspot mutants by peptide nucleic acid (PNA)-mediated PCR clamping. It markedly reduces the labor and cost of this kind of studies.

Oligonucleotide probes for Bordetella bronchiseptica based on 16S ribosomal RNA sequences

Bordetella bronchiseptica 16S ribosomal RNA (rRNA) gene was cloned and identified. On the basis of information from computer-assisted sequence comparison of the B. bronchiseptica 16S RRNA sequences with that of other bacterial species, we constructed B. bronchiseptica-specific oligonucleotide probes complementary to variable regions in the 16S rRNA molecule. Specificity of these 32P-labeled oligo-nucleotide probes was tested in a RNA/DNA hybridization with B. bronchiseptica strains and other bacterial strains. Probe BB4 was more specific than three other oligonucleotide probes. This probe BB4 was sensitive enough to be able to detect 10(4) bacterial cells.