Atsushi Kitayama

Global gene expression profiling and cluster analysis in Xenopus laevis.

We have undertaken a large-scale microarray gene expression analysis using cDNAs corresponding to 21,000 Xenopus laevis ESTs. mRNAs from 37 samples, including embryos and adult organs, were profiled. Cluster analysis of embryos of different stages was carried out and revealed expected affinities between gastrulae and neurulae, as well as between advanced neurulae and tadpoles, while egg and feeding larvae were clearly separated. Cluster analysis of adult organs showed some unexpected tissue-relatedness, e.g. kidney is more related to endodermal than to mesodermal tissues and the brain is separated from other neuroectodermal derivatives. Cluster analysis of genes revealed major phases of co-ordinate gene expression between egg and adult stages. During the maternal-early embryonic phase, genes maintaining a rapidly dividing cell state are predominantly expressed (cell cycle regulators, chromatin proteins). Genes involved in protein biosynthesis are progressively induced from mid-embryogenesis onwards. The larval-adult phase is characterised by expression of genes involved in metabolism and terminal differentiation. Thirteen potential synexpression groups were identified, which encompass components of diverse molecular processes or supra-molecular structures, including chromatin, RNA processing and nucleolar function, cell cycle, respiratory chain/Krebs cycle, protein biosynthesis, endoplasmic reticulum, vesicle transport, synaptic vesicle, microtubule, intermediate filament, epithelial proteins and collagen. Data filtering identified genes with potential stage-, region- and organ-specific expression. The dataset was assembled in the iChip microarray database, , which allows user-defined queries. The study provides insights into the higher order of vertebrate gene expression, identifies synexpression groups and marker genes, and makes predictions for the biological role of numerous uncharacterized genes.

Global analysis of RAR‐responsive genes in the Xenopus neurula using cDNA microarrays

Retinoid signaling is important for patterning the vertebrate hindbrain and midaxial regions. We recently showed that signaling through retinoic acid receptors (RARs) is essential for anteroposterior patterning along the entire body axis. To further investigate the mechanisms through which RARs act, we used microarray analysis to investigate the effects of modulating RAR activity on target gene expression. We identified 334 up‐regulated genes (92% of which were validated), including known RA‐responsive genes, known genes that have never been proposed as RA targets and many hypothetical and unidentified genes (n = 166). Sixty‐seven validated down‐regulated genes were identified, including known RA‐responsive genes and anterior marker genes. The expression patterns of selected up‐regulated genes (n = 45) were examined at neurula stages using whole‐mount in situ hybridization. We found that most of these genes were expressed in the neural tube and many were expressed in anterior tissues such as neural crest, brain, eye anlagen, and cement gland. Some were expressed in tissues such as notochord, somites, pronephros, and blood islands, where retinoic acid (RA) plays established roles in organogenesis. Members of this set of newly identified RAR target genes are likely to play important roles in neural patterning and organogenesis under the control of RAR signaling pathways, and their further characterization will expand our understanding of RA signaling during development. Developmental Dynamics 232:414–431, 2005.

Microbial ecology of nitrifying bacteria in wastewater treatment process examined by fluorescence in situ hybridization

The microbial ecology of nitrifying bacteria in various types of wastewater treatment processes and the dynamic response of the microbial ecology in biofilms were investigated using fluorescence in situ hybridization (FISH) with 16S rRNA-targeted oligonucleotide probes. Nitrifying bacteria were found to exhibit various organizational forms under different conditions of substrate composition and concentration. Ammonia-oxidizing bacteria were dominant in ammonia-rich inorganic wastewater, while heterotrophic bacteria and ammonia-oxidizing bacteria were localized at different positions in the biofilm in organic wastewater. The dynamics of the microbial ecology in the biofilm with regard to the spatial distribution of ammonia-oxidizing bacteria and heterotrophic bacteria caused by a gradual change in substrate composition was successfully monitored by FISH analysis.

Creation of a Thermostable Firefly Luciferase with pH-insensitive Luminescent Color¶

The thermal instability and pH‐sensitive spectral property of firefly luciferase have hampered its use as a sensitive multicolor luminescent label or bioluminescent resonance energy transfer donor. With the intention of improving the thermostability of a previously found firefly Hotaria parvula luciferase mutant with minor pH‐sensitive spectral change (V368A), further mutation (E356R) was introduced by taking a reportedly stabilized mutant of Photinus pyralis luciferase into account. The double mutant E356R/V368A showed significantly improved thermostability because >90% activity remained after incubation for 1 h at 45°C, with its specific activity being maintained. Unlike the wild type or V368A, E356R/V368A showed no change in the emission maximum of 568 nm even at pH 6.3, also implying a mutual relationship between thermostability and the proportion of yellow–green luminescent peak under acidic condition.

Detection of protein-protein interaction by bioluminescence resonance energy transfer from firefly luciferase to red fluorescent protein

We describe a novel homogeneous protein-protein interaction assay based on bioluminescence resonance energy transfer (BRET) from mutant firefly luciferase to red fluorescent protein (DsRed). The association of glutathione S-transferase (GST)-luciferase and protein G-DsRed, which was driven by the addition of anti-GST antibody, was successfully monitored by BRET.

Macroarray-based analysis of tail regeneration in Xenopus laevis larvae

Xenopus larvae possess a remarkable ability to regenerate their tails after they have been severed. To gain an understanding of the molecular mechanisms underlying tail regeneration, we performed a cDNA macroarray‐based analysis of gene expression. A Xenopus cDNA macroarray representing 42,240 independent clones was differentially hybridized with probes synthesized from the total RNA of normal and regenerating tails. Temporal expression analysis revealed that the up‐regulated genes could be grouped into early or late responding genes. A comparative expression analysis revealed that most genes showed similar expression patterns between tail development and regeneration. However, some genes showed regeneration‐specific expression. Finally, we identified 48 up‐regulated genes that fell into several categories based on their putative functions. These categories reflect the various processes that take place during regeneration, such as inflammation response, wound healing, cell proliferation, cell differentiation, and control of cell structure. Thus, we have identified a panel of genes that appear to be involved in the process of regeneration. Developmental Dynamics 233:1394–1404, 2005.

Screening of FGF target genes in Xenopus by microarray: temporal dissection of the signalling pathway using a chemical inhibitor

Microarray is a powerful tool for analysing gene expression patterns in genome‐wide view and has greatly contributed to our understanding of spatiotemporal embryonic development at the molecular level. Members of FGF (fibroblast growth factor) family play important roles in embryogenesis, e.g. in organogenesis, proliferation, differentiation, cell migration, angiogenesis, and wound healing. To dissect spatiotemporally the versatile roles of FGF during embryogenesis, we profiled gene expression in Xenopus embryo explants treated with SU5402, a chemical inhibitor specific to FGF receptor 1 (FGFR1), by microarray. We identified 38 genes that were down‐regulated and 5 that were up‐regulated in response to SU5402 treatment from stage 10.5–11.5 and confirmed their FGF‐dependent transcription with RT‐PCR analysis and whole‐mount in situ hybridization (WISH). Among the 43 genes, we identified 26 as encoding novel proteins and investigated their spatial expression pattern by WISH. Genes whose expression patterns were similar to FGFR1 were further analysed to test whether any of them represented functional FGF target molecules. Here, we report two interesting genes: one is a component of the canonical Ras‐MAPK pathway, similar to mammalian mig6 (mitogen‐inducible gene 6) acting in muscle differentiation; the other, similar to GPCR4 (G‐protein coupled receptor 4), is a promising candidate for a gastrulation movement regulator. These results demonstrate that our approach is a promising strategy for scanning the genes that are essential for the regulation of a diverse array of developmental processes.

Flow injection microbial trichloroethylene sensor

A flow type microbial biosensor for direct measurement of trichloroethylene (TCE) was developed. The unique features of this novel microbial sensor were the use of the TCE degrading bacterium Pseudomonas aeruginosa JI104, the electrical detection of the chloride ion released by microbial degradation, and flow cell made of glass. Glass cell was used in order to suppress adsorption of TCE and made a closed reaction cell. Vaporization of TCE during the measurement was prevented using closed flow cell. The performance of the sensor was evaluated from following aspects; such as pH of the carrier solution, amount of the immobilized microbe, flow rate and injection volume. The sensor signals were linearly proportional to the concentration of TCE in the range from 0.03 to 2 mgl(-1), which is suitable for the determination of suspected samples to be drinkable water or not. The sensor performance was checked on the real sample, and this system showed good response in ground water, indicating its applicability for the on line monitoring at TCE contaminated areas or hazardous sites.

Gene organization and low regiospecificity in aromatic-ring hydroxylation of a benzene monooxygenase of Pseudomonas aeruginosa JI104

Abstract A novel benzene monooxygenase gene cluster has been cloned from Pseudomonas aeruginosa JI104 which was isolated from soil as a benzene degrader. The nucleotide sequence of this gene cluster was found to be highly homologous to those of other toluene monooxygenase gene clusters. This multicomponent monooxygenase also has the capability to catalyze the hydroxylation of various alkylated aromatic hydrocarbons. The low regiospecific hydroxylation was observed when toluene, o-xylene, ethyl benzene and n-propyl benzene were used as substrates.

Identification of neural genes using Xenopus DNA microarrays

To isolate novel genes regulating neural induction, we used a DNA microarray approach. As neural induction is thought to occur by means of the inhibition of bone morphogenetic protein (BMP) signaling, BMP signaling was inhibited in ectodermal cells by overexpression of a dominant‐negative receptor. RNAs were isolated from control animal cap explants and from dominant‐negative BMP receptor expressing animal caps and subjected to a microarray experiment using newly generated high‐density Xenopus DNA microarray chips representing over 17,000 unigenes. We have identified 77 genes that are induced in animal caps after inhibition of BMP signaling, and all of these genes were subjected to whole‐mount in situ hybridization analysis. Thirty‐two genes showed specific expression in neural tissues. Of the 32, 14 genes have never been linked to neural induction. Two genes that are highly induced by BMP inhibition are inhibitors of Wnt signaling, suggesting that a key step in neural induction is to produce Wnt antagonists to promote anterior neural plate development. Our current analysis also proves that a microarray approach is useful in identifying novel candidate factors involved in neural induction and patterning. Developmental Dynamics 232:432–444, 2005.