Rapid and Sensitive Assay of K-ras Mutations in Pancreatic Cancer by Electrochemical Detection with Ferrocenyl-naphthalene-diimide.

The DNA chip is a very powerful tool for genetic analysis. Conventional DNA chips that utilize fluorescence detection systems are very complicated, expensive and impractical, but the electrochemical array (ECA) chip is gaining popularity. To investigate the validity of the ECA chip, which utilizes ferrocenyl-naphthalene-diimide (FND), k-ras mutations in 20 pancreatic cancer tissues were examined. DNA was isolated from 20 pancreatic cancer tissues and subjected to a 2-stage polymerase chain reaction (PCR). The k-ras mutations were detected with the ECA chip. To verify the reliability of the ECA chip, the DNA was also analyzed by direct sequencing and the PCR-dependent preferential homoduplex formation assay (PCR-PHFA). The ECA chip could detect one mutation in a background of 1000 wild-type DNAs. K-ras mutations were identified in 17 out of 20 (85%) pancreatic cancer samples. Three mutations of codon 12 of k-ras, GTT, GAT and AGT, were detected. K-ras mutations were detected in 13 out of 20 (65%) samples by sequencing and in 17 out of 20 (85%) samples by PCR-PHFA. These findings were concordant with the ECA chip result. The FND-ECA chip is a sensitive, rapid and reliable method for screening point mutations in a variety of clinical samples.

A novel method of identifying genetic mutations using an electrochemical DNA array.

We describe the development of a new type of DNA array chip that utilizes electrochemical reactions and a novel method of simultaneously identifying multiple genetic mutations on an array chip. The electrochemical array (ECA) uses a threading intercalator specific to double-stranded nucleotides, ferrocenylnaphthalene diimide (FND), as the indicator. ECA does not require target labeling, and the equipment is simple, durable and less expensive. The simultaneous multiple mutation detection (SMMD) system using an ECA chip and FND utilizes an enzyme to simultaneously distinguish several genetic mutations such as single nucleotide polymorphism (SNP), insertion, deletion, translocation and short tandem repeat. We examined this SMMD system using an ECA chip, by detecting seven different mutations on the lipoprotein lipase (LPL) gene for 50 patients in a blind test. It turned out that all the results obtained were concordant with the sequencing results, demonstrating that this system is a powerful tool for clinical applications.

An Ets transcription factor, HrEts, is target of FGF signaling and involved in induction of notochord, mesenchyme, and brain in ascidian embryos.

In ascidian embryos, a fibroblast growth factor (FGF) signal induces notochord, mesenchyme, and brain formation. Although a conserved Ras/MAPK pathway is known to be involved in this signaling, the target transcription factor of this signaling cascade has remained unknown. We have isolated HrEts, an ascidian homolog of vertebrate Ets1 and Ets2, to elucidate the transcription factor involved in the FGF signaling pathway in embryos of the ascidian Halocynthia roretzi. Maternal mRNA of HrEts was detected throughout the entire egg cytoplasm and early embryos. Its zygotic expression started in several tissues, including the notochord and neural plate. Overexpression of HrEts mRNA did not affect the general organization of the tadpoles, but resulted in formation of excess sensory pigment cells. In contrast, suppression of HrEts function by morpholino antisense oligonucleotide resulted in severe abnormalities, similar to those of embryos in which the FGF signaling pathway was inhibited. Notochord-specific Brachyury expression at cleavage stage and notochord differentiation at the tailbud stage were abrogated. Formation of mesenchyme cells was also suppressed, and the mesenchyme precursors assumed muscle fate. In addition, expression of Otx in brain-lineage blastomeres was specifically suppressed. These results suggest that an Ets transcription factor, HrEts, is involved in signal transduction of FGF commonly in notochord, mesenchyme, and brain induction in ascidian embryos.

Expression pattern and transcriptional control of SoxB1 in embryos of the ascidian Halocynthia roretzi.

The Sox family is a large group of transcription factors that are characterized by the presence of a DNA-binding HMG domain. We isolated HrSoxB1, an ascidian homolog of the Sox gene that belongs to the B1 subclass of the Sox family, from Halocynthia roretzi. Expression was initiated as early as the 8-cell stage. During cleavage stages, HrSoxB1 was expressed in three quarters of embryonic blastomeres but not in posterior-vegetal (B-line) blastomeres. Misexpression of mRNAs of HrPEM but not of macho-1, whose maternal mRNAs are localized to the posterior-vegetal cytoplasm of eggs and early embryos, repressed the anterior-vegetal expression of HrSoxB1. This result suggests that the zygotic expression of HrSoxB1 is controlled by the localized maternal mRNA. When HrSoxB1 was overexpressed in early embryos, ectopic expression of HrBra, a gene for a transcription factor expressed in notochord blastomeres, occurred in the most posterior blastomeres (B7.5), although these blastomeres did not eventually differentiate into notochord but developed into muscle, as they do in normal embryogenesis. In later embryogenesis, HrSoxB1 was specifically expressed in neural plate cells. However, overexpression of HrSoxB1 did not affect the expression of a neural plate marker gene, HrETR-1.

Repression of zygotic gene expression in the putative germline cells in ascidian embryos.

In germline cells of early C. elegans and Drosophila embryos, repression of zygotic gene expression appears to be essential to maintain the germ cell fate. In these animals, specific residues in the carboxy-terminal domain (CTD) of RNA polymerase II large subunit (RNAP II LS) are dephosphorylated in the germline cells, whereas they are phosphorylated in the somatic cells. We investigated, in early embryos of the ascidian Halocynthia roretzi, the expression patterns of three genes that are essentially expressed in the entire embryo after the 32-cell stage. We found that the expression of these genes was inactive in the putative germline cells during the cleavage stages. Once cells were separated from the germline lineage by cleavages, the expression of the genes was initiated in the cells. These results suggest that repression of transcription in germline cells may also be common in chordate embryos. We then examined the phosphorylation state of the CTD of RNAP II using a phosphoepitope-specific antibody. At cleavage stages after the 32-cell stage, CTD was phosphorylated in every blastomere, including the germline cells. Therefore, in the ascidian, the inactivation of zygotic transcription is not correlated with dephosphorylation of the CTD. These observations indicate that zygotic transcription is inactivated in ascidian germline cells, but the mechanism of the repression may differ from that in C. elegans and Drosophila.

Isolation of cDNA clones for mRNAs transcribed zygotically during cleavage in the ascidian, Halocynthia roretzi.

The ascidian larva consists of a relatively small number of different cell types, and the cell lineages during embryogenesis have been well described. The clonal restriction of developmental fate takes place considerably early in development. The fates of most of the blastomeres become tissue-restricted by the 110-cell stage, just before the onset of gastrulation. To elucidate the molecular basis of the early events of fate determination in the ascidian Halocynthia roretzi, we isolated the genes for which zygotic expression is initiated during the early cleavage stages. Here we report 18 genes isolated by subtractive hybridization screening between 110-cell embryos and fertilized eggs. The expression of most (13) of the genes was initiated at the 32-cell stage. The genes were subdivided into three groups according to their spatial expression patterns. The first group included clones expressed throughout almost the entire embryo. The second and third groups represented clones expressed mainly in the animal hemisphere and in a subset of vegetal blastomeres, respectively. One of the genes, HrHesl1, encoded a polypeptide containing the bHLH domain that is similar to those of the Hairy/Enhancer of split/Deadpan family of transcriptional repressors. HrHesl1 was expressed exclusively in epidermal precursor cells during cleavage. Another gene named HrWnt-5 beta was expressed in muscle precursors.

Expression of a Gene for Major Mitochondrial Protein, ADP/ATP Translocase, during Embryogenesis in the Ascidian Halocynthia roretzi: (Ascidian embryos/ADP/ATP translocase gene/maternal mRNA/mitochondria).

The ADP/ATP translocase is the most abundant integral protein of the inner mitochondrial membrane and it is encoded by the nuclear DNA. Because mitochondria in the ascidian egg appear to be segregated into blastomeres of muscle lineage, we examined the expression of a gene for ADP/ATP translocase during embryogenesis of the ascidian Halocynthia roretzi. Sequence analysis of a cDNA clone for the ascidian ADP/ATP translocase indicated that it contains a single open reading frame that encodes a polypeptide of 304 amino acids. The polypeptide showed extensive similarity to mammalian ADP/ATP translocases, with as much as 74% identity. The genome of H. roretzi contains a single gene, or two genes at most, for the protein. A large amount of maternal mRNA for ADP/ATP translocase was found in unfertilized eggs and early embryos. The amount of this mRNA decreased after the 64-cell stage, and the mRNA became barely detectable in tailbud embryos, although zygotic transcript for the protein was evident in adult tissues. Both in situ hybridization and Northern blot analyses demonstrated that the mRNA is distributed in the entire cytoplasm of unfertilized eggs. The mRNA is segregated during embryogenesis not only into blastomeres of muscle lineage but also into those of non-muscle lineage.