Adapter-tagged competitive PCR (ATAC-PCR)--a high-throughput quantitative PCR method for microarray validation

Adapter-tagged competitive PCR (ATAC-PCR) is an advanced version of competitive quantitative PCR that is characterized by the addition of unique adapters to cDNA derived from each sample RNA. Using multiple adapters, we can accurately measure the relative expression ratios of many samples, with a calibration curve obtained from internal standards included in the same reaction. ATAC-PCR can identify differences in gene expression as small as twofold, even from very small amounts of sample RNA. This technique is suitable for confirming results obtained with cDNA microarrays or differential display, and it can process more than a thousand of genes per day when used in conjunction with a capillary DNA sequencer.     

Genetic engineering of Escherichia coli for production of tetrahydrobiopterin

Tetrahydrobiopterin (BH4) is an essential cofactor for various enzymes in mammals. In vivo, it is synthesized from GTP via the three-step pathway of GTP cyclohydrolase I (GCHI), 6-pyruvoyl-tetrahydropterin synthase (PTPS) and sepiapterin reductase (SPR). BH4 is a medicine used to treat atypical hyperphenylalaninemia. It is currently synthesized by chemical means, which consists of many steps, and requires costly materials and complicated procedures. To explore an alternative microbial method for BH4 production, we utilized recombinant DNA technology to construct recombinant Escherichia coli (E. coli) strains carrying genes expressing GCHI, PTPS and SPR enzymes. These strains successfully produced BH4, which was detected as dihydrobiopterin and biopterin, oxidation products of BH4. In order to increase BH4 productivity we made further improvements. First, to increase the de novo GTP supply, an 8-azaguanine resistant mutant was isolated and an additional guaBA operon was introduced. Second, to augment the activity of GCHI, the folE gene from E. coli was replaced by the mtrA gene from Bacillus subtilis. These modifications provided us with a strain showing significantly higher productivity, up to 4.0 g of biopterin/L of culture broth. The results suggest the possibility of commercial BH4 production by our method.     

A novel DNA polymerase homologous to Escherichia coli DNA polymerase I from a higher plant, rice (Oryza sativa L.)

A novel DNA polymerase, designated as OsPolI-like, has been identified from the higher plant, rice (Oryza sativa L. cv. Nipponbare). The OsPolI-like cDNA was 3765 bp in length, and the open reading frame encoded a predicted product of 977 amino acid residues with a molecular weight of 100 kDa. The OsPolI-like gene has been mapped to chromosome 8 and contains 12 exons and 11 introns. The encoded protein showed a high degree of sequence and structural homology to Escherichia coli pol I protein, but differed from DNA polymerase γ and θ. The DNA polymerase domain of OsPolI-like showed DNA polymerase activity. Subcellular fractionation analysis suggested that the protein is localized in the plastid. Northern and western blotting, and in situ hybridization analyses demonstrated preferential expression of OsPolI-like in meristematic tissues such as shoot apical meristem, root apical meristem, leaf primordia and the marginal meristem. Interestingly, no expression was detected in mature leaves, although they have a high chloroplast content. These properties indicated that OsPolI-like is a novel plant DNA polymerase. The function of OsPolI-like is discussed in relation to plastid maturation.     

Structural and functional diversities of the Aplysia Mytilus inhibitory peptide-related peptides

Aplysia Mytilus inhibitory peptide-related peptides (AMRPs) are multiple hexapeptides coded on a single precursor. By comparing the AMRP precursors of two species of Aplysia (Aplysia californica and Aplysia kurodai), we found that there are substantial numbers of species-specific AMRPs. We next compared the function of AMRPs on the anterior aorta between A. kurodai and Aplysia juliana. In A. juliana, AMRPs inhibited the contractile activity of the aorta (EC(50)=10(-9) to 10(-8)M), whereas the peptides had no obvious action in A. kurodai up to 10(-7)M. These results indicate that AMRPs are both structurally and functionally diverse neuropeptides even among closely related species.     

Aplysia cardioactive peptide (NdWFamide) enhances the L-type Ca2+ current of Aplysia ventricular myocytes

NdWFamide is a D-amino acid containing tripeptide purified from Aplysia heart. Although the cardioexcitatory action of NdWFamide is well established, little is known about how the excitatory action is induced. To examine the action of the peptide on the ion channels expressed in the Aplysia heart muscles, we carried out whole cell clamp experiments in the isolated Aplysia ventricular myocytes. We found that the high voltage-activated (HVA) Ca(2+) current of Aplysia ventricular myocytes is mostly a nifedipine-sensitive L-type current, and that the current was enhanced by NdWFamide via the activation of G proteins.     

A novel GGNG-related neuropeptide from the polychaete Perinereis vancaurica

The GGNG peptides are myoactive peptides so far identified from earthworms and leeches, which are the earthworm excitatory peptides (EEP) and the leech excitatory peptide (LEP), respectively. A novel GGNG peptide was isolated and structurally determined from a marine polychaete, Perinereis vancaurica, using a combination of immunological assay and high performance liquid chromatography (HPLC). The peptide was a pentadecapeptide whose amino acid sequence was similar to that of EEP and LEP, and showed myoactivity on isolated esophagus of P. vancaurica with a threshold concentration of 10(-10)M. The peptide was designated as polychaete excitatory peptide (PEP). Amidation of the alpha-carboxyl group of C-terminal residue occurred in PEP. This is the case for LEP, but not for EEP. The cDNA cloning revealed that the structure of the PEP precursor is more similar to the EEP precursor than to the LEP precursor. Immunohistochemical staining showed the presence of PEP in several neurons of central nervous system (CNS) as somata and neuropile structure, epithelial cells of the pharynx and epidermal cells throughout the body wall. Altogether these results support the physiological significance of PEP in regulation of the CNS neural activity and the peripheral myoactivity.     

Conjugation of various acridines to DNA for site-selective RNA scission by lanthanide ion

Three types of DNA conjugates having 9-acridinecarboxamide, 9-aminoacridine, and 9-amino-6-chloro-2-methoxyacridine at the 5'-ends were synthesized and used for site-selective RNA scission together with another unmodified DNA and Lu(III) ion. The target phosphodiester linkages in the substrate RNA were selectively and efficiently activated and were hydrolyzed by free Lu(III) ion. The conjugate bearing 9-amino-6-chloro-2-methoxyacridine was the most active. However, its duplex with the substrate RNA was almost as stable as that of the 9-aminoacridine-bearing conjugate, which was much less active for the RNA activation. The 9-acridinecarboxamide-bearing conjugate was only marginally active. The substituents on the acridine groups in these conjugates positively participate in the present RNA activation, probably by fixing the orientation of the acridine rings.     

Ligand-independent dimer formation of epidermal growth factor receptor (EGFR) is a step separable from ligand-induced EGFR signaling

Dimerization and phosphorylation of the epidermal growth factor (EGF) receptor (EGFR) are the initial and essential events of EGF-induced signal transduction. However, the mechanism by which EGFR ligands induce dimerization and phosphorylation is not fully understood. Here, we demonstrate that EGFRs can form dimers on the cell surface independent of ligand binding. However, a chimeric receptor, comprising the extracellular and transmembrane domains of EGFR and the cytoplasmic domain of the erythropoietin receptor (EpoR), did not form a dimer in the absence of ligands, suggesting that the cytoplasmic domain of EGFR is important for predimer formation. Analysis of deletion mutants of EGFR showed that the region between (835)Ala and (918)Asp of the EGFR cytoplasmic domain is required for EGFR predimer formation. In contrast to wild-type EGFR ligands, a mutant form of heparin-binding EGF-like growth factor (HB2) did not induce dimerization of the EGFR-EpoR chimeric receptor and therefore failed to activate the chimeric receptor. However, when the dimerization was induced by a monoclonal antibody to EGFR, HB2 could activate the chimeric receptor. These results indicate that EGFR can form a ligand-independent inactive dimer and that receptor dimerization and activation are mechanistically distinct and separable events.