Wei-yuan Chow | National Tsing Hua University (original) (raw)

Papers by Wei-yuan Chow

Biological research topics gradually shift from structural genomics into functional genomics. DNA... more Biological research topics gradually shift from structural genomics into functional genomics. DNA microarrays have been used to generate abundant data for exploring functions and interactions among genes. We propose a reverse-engineering strategy to predict the interactions between genes within a genetic network. Our inputs are perturbation matrices experimentally obtained from DNA microarrays. First, we make some assumptions for the interactions in the network. The proposed network is represented as a directed graph. After that, we enumerate all possible network models according to the assumptions. And then, some candidate models are obtained, resulted from calculated perturbation matrices out of computational simulation. The network involves in not only the transcription level but also the nucleotide/protein interactions in general. To justify this method, we take a well-known genetic regulatory network in yeast Saccharomyces cerevisia for a test. The result shows that one of the ...

Nucleic acids research, 2006

The identification of regulatory elements recognized by transcription factors and chromatin remod... more The identification of regulatory elements recognized by transcription factors and chromatin remodeling factors is essential to studying the regulation of gene expression. When no auxiliary data, such as orthologous sequences or expression profiles, are used, the accuracy of most tools for motif discovery is strongly influenced by the motif degeneracy and the lengths of sequence. Since suitable auxiliary data may not always be available, more work must be conducted to enhance tool performance to identify transcription elements in the metazoan. A non-alignment-based algorithm, MotifSeeker, is proposed to enhance the accuracy of discovering degenerate motifs. MotifSeeker utilizes the property that variable sites of transcription elements are usually position-specific to reduce exposure to noise. Consequently, the efficiency and accuracy of motif identification are improved. Using data fusion, the ranking process integrates two measures of motif significance, resulting in a more robust ...

Molecular and Cellular Neuroscience, 2014

Local synthesis of proteins in the axons participates in axonogenesis and axon guidance to establ... more Local synthesis of proteins in the axons participates in axonogenesis and axon guidance to establish appropriate synaptic connections and confer plasticity. To study the transcripts present in the growth cones and axonal shafts of cultured rat hippocampal neurons, two chip devices, differing in their abilities to support axonal growth and branching, are designed and employed here to isolate large quantities of axonal materials. Cone-, shaft- and axon-residing transcripts with amounts higher than that of a somatodendritic transcript, Actg1 (γ-actin), are selected and classified. Since the chips are optically transparent, distribution of transcripts over axons can be studied by fluorescence in situ hybridization. Three transcripts, Cadm1 (cell adhesion molecule 1), Nefl (neurofilament light polypeptide), and Cfl1 (non-muscle cofilin) are confirmed to be preferentially localized to the growth cones, while Pfn2 (profilin2) is preferentially localized to the shafts of those axons growing on the chip that restricts axonal growth. The different growing conditions of axons on chips and on conventional coverslips do not affect the cone-preferred localization of Cadm1 and shaft-preferred localization of Pfn2, but affect the distributions of Nefl and Cfl1 over the axons at 14th day in vitro. Furthermore, the distributions of Cadm1 and Nefl over the axons growing on conventional coverslips undergo changes during in vitro development. Our results suggest a dynamic nature of the mechanisms regulating the distributions of transcripts in axonal substructures in a manner dependent upon both growth conditions and neuronal maturation.

Nucleic Acids Research, 2006

GeneAlign is a coding exon prediction tool for predicting protein coding genes by measuring the h... more GeneAlign is a coding exon prediction tool for predicting protein coding genes by measuring the homologies between a sequence of a genome and related sequences, which have been annotated, of other genomes. Identifying protein coding genes is one of most important tasks in newly sequenced genomes. With increasing numbers of gene annotations verified by experiments, it is feasible to identify genes in the newly sequenced genomes by comparing to annotated genes of phylogenetically close organisms. GeneAlign applies CORAL, a heuristic linear time alignment tool, to determine if regions flanked by the candidate signals (initiation codon-GT, AG-GT and AG-STOP codon) are similar to annotated coding exons. Employing the conservation of gene structures and sequence homologies between protein coding regions increases the prediction accuracy. GeneAlign was tested on Projector dataset of 491 human-mouse homologous sequence pairs. At the gene level, both the average sensitivity and the average specificity of GeneAlign are 81%, and they are larger than 96% at the exon level. The rates of missing exons and wrong exons are smaller than 1%. GeneAlign is a free tool available at http://

Lab on a Chip, 2010

Axons are long, slender processes extending from the cell bodies of neurons and play diverse and ... more Axons are long, slender processes extending from the cell bodies of neurons and play diverse and crucial roles in the development and function of nervous systems. Here, we describe the development of a chip device that can be used to produce large quantities of axons for proteomic and RNA analyses. On the chip surface, bundles of axons of rat hippocampal neurons in culture are guided to grow in areas distinct and distant from where their cell bodies reside. Fluorescence immunocytochemical studies have confirmed that the areas where these axons are guided to grow are occupied exclusively by axons and not by neuronal somatodendrites or astroglial cells. These axon-occupied parts are easily separated from the remainder of the chip and collected by breaking the chip along the well-positioned linear grooves made on the backside. One-and two-dimensional gel electrophoresis and Western blotting analyses reveal that the axons and whole cells differ in their protein compositions. RT-PCR analyses also indicate that the axons contain only a subset of neuronal RNAs. Furthermore, the chip device could be easily modified to address other issues concerning neuronal axons, such as the molecular composition of the axon substructure, the growth cone and shaft, the degeneration and regeneration processes associated with injured axons and the effects of extrinsic molecules, such as axon guidance cues and cell adhesion molecules, on the axon. With these diverse applications, the chip device described here will serve as a powerful platform for studying the functional proteome of neuronal axons.

Journal of Neuroscience Research, 2009

Dendritic spines are small protrusions on neuronal dendrites and the major target of the excitato... more Dendritic spines are small protrusions on neuronal dendrites and the major target of the excitatory inputs in mammalian brains. Cultured neurons and brain slices are important tools in studying the biochemical and cellular properties of dendritic spines. During the processes of immunocytochemical studies of neurons and the preparation of brain slices, neurons were often kept at temperatures lower than 378C for varied lengths of time. This study sought to investigate whether and how cold treatment would affect the protein composition of dendritic spines. The results indicated that upon cold treatment four postsynaptic proteins, namely, a,b-tubulins, calcium, calmodulin-dependent protein kinase IIa, and cytoplasmic dynein heavy chain and microtubuleassociated protein 2, but not PSD-95 or AMPA receptors, exited from the majority of dendritic spines of cultured rat hippocampal neurons in a Gd 31 -sensitive manner. The cold-induced exit of tubulins from dendritic spines was further found to be an energy-dependent process involving the activation of Gd 31 -sensitive calcium channels and ryanodine receptors. The results thus indicate that changes in temperature, calcium concentration, and energy supply of the medium surrounding neurons would affect the protein composition of the dendritic spines and conceivably the protein composition of the subcellular organizations, such as the postsynaptic density, in the cytoplasm of dendritic spines. V V C 2008 Wiley-Liss, Inc.

Journal of Neurochemistry, 2002

We describe here the isolation and biochemical characterization of a population of protein aggreg... more We describe here the isolation and biochemical characterization of a population of protein aggregates from the postsynaptic density (PSD) prepared from pig cerebral cortex. The protein constituents of these aggregates are linked together primarily by disulfide bonds. Negative staining electron microscopy revealed that the isolated protein aggregates were granular objects with an average outside diameter of approximately 21 nm and with small protrusions on their surface. The major constituents of the isolated granular aggregates consist of tubulin and an unidentified protein of 70 kDa in size. Small amounts of the alpha subunit of calcium/calmodulin-dependent protein kinase II and subunits of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid and NMDA subtypes of glutamate receptors were also detected by immunoblotting. Actin, however, was not found in these granular aggregates. We propose that these granular protein aggregates correspond to the approximately 20-nm-diameter granular particles of the PSD on the basis of their biochemical and morphological characteristics. The spatial arrangement of these granular aggregates relative to other components of the postsynaptic terminal is also postulated here.

Journal of Molecular Evolution, 2001

The AMPA receptor (AMPAR), a pharmacologically defined ionotropic glutamate receptor, mediates fa... more The AMPA receptor (AMPAR), a pharmacologically defined ionotropic glutamate receptor, mediates fast excitatory synaptic transmission in the vertebrate central nervous system. Mammalian and avian AMPARs are assembled from the products of four genes (GRIA1-GRIA4) conserved in their translated sequences and gene organizations. Teleost fish also express AMPAR subunits; however, the AMPAR genes have not been extensively investigated in lower vertebrates. To elucidate the evolution of vertebrate AMPAR genes, reverse-transcriptase PCR-based surveys of subunits expressed in the brains of eight nonmammalian vertebrates were performed. The newly cloned vertebrate AMPAR subunits were classified by their sequence identities to the mammalian AMPAR subunits. The results of molecular and phylogenetic analyses indicated that the members of the AMPAR gene family increased from two in the jawless hagfish to four in the tetrapods and the shark and to more than four in the teleost fish. The sizes of AMPAR gene families correlate well with those of many multigene families observed in various vertebrates. Moreover, all vertebrates expressed at least one AMPAR subunit bearing an arginine (R) at the Q/R site, at which no invertebrate glutamate receptor subunit has been found to have an R residue, suggesting that the low calcium-permeable AMPARs appeared at early evolutionary stages of vertebrate central nervous systems.

Journal of Molecular Evolution, 2006

The incomplete correlation between the organismal complexities and the number of genes among euka... more The incomplete correlation between the organismal complexities and the number of genes among eukaryotic organisms can be partially explained by multiple protein products of a gene created by alternative splicing. One type of alternative splicing involves alternative selection of mutually exclusive exons and creates protein products with substitution of one segment of the amino acid sequence for another. To elucidate the evolution of the mutually exclusive 115-bp exons, designated flip and flop, of vertebrate AMPA receptor genes, the gene structures of chordate (tunicate, cephalochordate, and vertebrate) and protostome (Drosophila and Caenorhabditis elegans) AMPA receptor subunits were compared. Phylogenetic analysis supports that the vertebrate flip and flop exons evolved from a common sequence. Flip and flop exons exist in all vertebrate AMPA receptor genes but only one 115-bp exon is present in the genes of tunicates and cephalochordates, suggesting that the exon duplication event occurred at the ancestral vertebrate AMPA receptor gene after the separation of vertebrates from primitive chordates. The structures of animal AMPA receptor genes also suggest that an intron insertion to separate the primordial flip/flop exon from the M4coding exon occurred before the exon duplication event and probably at the chordate lineage.

Gene, 1996

Metallothionein (MT) cDNAs were cloned and sequenced from two genera of ducks, Muscovy (Cairina m... more Metallothionein (MT) cDNAs were cloned and sequenced from two genera of ducks, Muscovy (Cairina muschata) and Tsai ya (Anas platyrhynchos). The two cDNAs show an extremely high sequence homology and contain an open reading frame encoding 63 amino acids. MT mRNA expressions were studied after metal induction using the cloned cDNA as a probe. Cadmium and copper induced MT gene efficiently, whereas zinc showed a markedly less effect. In addition, the MT mRNA accumulations in various developmental stages were also investigated. The result reveals a different pattern of expression from that of mammals. The discrepancy in MT gene between Tsai ya and Muscovy was further explored by examining genomic DNA structures. The duck MT showed three exons and two introns. The most significant variation of the genes occurs at intron II in which Tsai ya MT has 24 bases more than Muscovy MT. Moreover, MT expressions in the hybrids of Muscovy and Tsai ya were investigated using a reverse transcriptase-polymerase chain reaction. Those results demonstrated that parental MT genes are expressed in the hybrids after metal induction.

Gene, 1999

The AMPA (alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid)-preferring receptor is one o... more The AMPA (alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid)-preferring receptor is one of the pharmacologically defined ionotropic glutamate receptors, which mediate fast excitatory synaptic transmission in the central nervous system of vertebrates. Here, we report the mapping of the transcriptional start points and identification of the intron-exon boundaries of the teleost AMPA receptor subunit gene fGluR2 beta. fGluR2 beta and the mouse GluR2 share a similar genomic organization, having identical intron insertion sites and a large intron 2; however, fGluR2 beta has an extra exon encoding an alternate 5'-UTR. Results of RT-PCR and RNase protection analyses indicate that mature fish brain expresses two types of fGluR2 beta transcripts with different 5' ends. Transcriptions of these two fGluR2 beta transcripts started from two chromosomal regions separated by at least 10 kb. Only the transcript starting from the region more upstream on the chromosome was spliced. Moreover, transcript initiated from the downstream region was more abundant than that initiated from the upstream region.

FEBS Letters, 2001

The amino acid, either a glutamine (Q) or an arginine (R), at the Q/R site of the pore-lining seg... more The amino acid, either a glutamine (Q) or an arginine (R), at the Q/R site of the pore-lining segment (M2) of a vertebrate AMPA receptor subunit critically influences the properties of the receptor. The R codon of the mammalian AMPA receptor subunit 2 (GRIA2) transcript is not coded by the chromosomal sequence, but is created by posttranscriptional RNA editing activities. On the other hand, the R codons of some teleost GRIA2 homologs are coded by chromosomal sequences. To elucidate the evolution of the utilization of Q/R RNA editing in modifying vertebrate GRIA2 transcripts, the GRIA2 genes of five fish species and an amphibian were studied. The putative hagfish GRIA2 homolog (hfGRIA2) encodes an R codon, whereas shark and bullfrog GRIA2 genes specify a Q codon at the genomic Q/R site. All gnathostoma GRIA2 genes possess an intron splitting the coding regions of M2 and the third hydrophobic region (M3). The intronic components required for Q/R RNA editing are preserved in all the Q-coding vertebrate GRIA2 genes but are absent from the R-coding GRIA2 genes. Interestingly, the hfGRIA2 is intronless, suggesting that hfGRIA2 is unlikely evolved from a Q/R editing-competent gene. Results of this study suggest that modification of GRIA2 transcripts by Q/R editing is most likely acquired after the separation of the Agnatha and Gnathostome. ß

DNA and Cell Biology, 1996

In this study, we isolated two cDNA molecules encoding putative glutamate receptor subunits, fGlu... more In this study, we isolated two cDNA molecules encoding putative glutamate receptor subunits, fGluR1 alpha and fGluR1 beta, from an Oreochromis sp. brain cDNA library by hybridizing with the glutamate receptor cDNA, fGluR2 beta, of the same fish. The deduced amino acid sequence of the fGluR1 alpha consists of 908 residues with an 18-residue signal peptide and displays a sequence identity of 74% to the amino acid sequence of rat GluR1 subunit. Northern blotting indicates that the expression level of fGluR1 alpha in telencephalon is higher than that in optic tectum and cerebellum in adult fish brain. Reverse-transcriptase polymerase chain reaction and genomic analyses reveal the presence of variants created by alternative splicing at the flip-flop module and the carboxyl terminus of fGluR1 alpha transcripts. The amino acid sequence of fGluR1 alpha is unique in that it contains a glutamine-rich sequence inserted at the loop 1 (L1) between transmembrane domains 1 and 2. A second incomplete cDNA clone, designated fGluR1 beta, coding for a polypeptide showing sequence identity to the rat GluR1 and fGluR1 alpha was isolated from the same library. Insertion of a serine- and glutamine-rich sequence at the L1 was also detected in the translated sequence of fGluR1 beta.

Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 2007

NMDA (N-methyl-d-aspartate) receptor, a subclass of the ionotropic glutamate receptors, participa... more NMDA (N-methyl-d-aspartate) receptor, a subclass of the ionotropic glutamate receptors, participates in synaptic transmission and plays important roles in various higher brain functions in the vertebrate central nervous system. Here, we report the cloning of two NR1 subunits of tilapia (Oreochromis mossambicus). Phylogenetic analysis strongly supports that the two tilapia NR1 genes are paralogous, resulting from a gene duplication event in the teleost lineage. The electrophysiological and pharmacological properties of the tilapia NR1.2 subunit coexpressed with rat NR2B in the Xenopus oocytes are similar to that of the recombinant rat NR1/NR2B. Both tilapia NR1 transcripts are alternatively spliced at the N and C terminal coding regions. The C terminal exons, C1&amp;amp;amp;amp;amp;amp;amp;amp;amp;#39; and C1&amp;amp;amp;amp;amp;amp;amp;amp;amp;quot;, originally discovered in the knifefish NR1 gene, are present in the tNR1.1 but not in the tNR1.2. Majorities of the NR1 transcripts expressed in the tilapia and zebrafish brains do not include these alternative splice exons. The splicing patterns of NR1 transcripts differ in various brain subregions. The regional expression patterns of splice variants are not fully preserved between tilapia and zebrafish. Nevertheless, tectum opticum regions of teleost and rat express high levels of NR1 splicing variant with N1 cassette.

Molecular Brain Research, 1998

Here we report the cloning and functional analysis of a cDNA encoding a functional glutamate rece... more Here we report the cloning and functional analysis of a cDNA encoding a functional glutamate receptor subunit of Oreochromis sp., a freshwater teleost fish. The deduced amino acid sequence of this cDNA clone, fGluR3 alpha, displays the highest sequence identity to that of the mammalian GluR3 subunit. Results of quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) analysis indicated that the expression level of fGluR3 alpha in the cerebellum was much less than that in the telencephalon and optical lobe. Similar to its mammalian counterpart, variants of fGluR3 alpha were created by alternative splicing and RNA editing at the R/G site. The channel properties of homomeric fGluR3 alpha expressed in Xenopus oocytes were similar to those of the mammalian alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-preferring receptors. The rank order of agonist potency of the expressed fGluR3 alpha is AMPA > or = glutamate > or = quisqualate > domoate > or = kainate. This is the first functional glutamate receptor of teleost fish being demonstrated to be sensitive to AMPA. Furthermore, this study suggested a strong functional conservation of AMPA-preferring receptors in vertebrates.

Brain Research, 2006

The AMPA-preferring receptors (AMPARs) mediate rapid excitatory synaptic transmission in the cent... more The AMPA-preferring receptors (AMPARs) mediate rapid excitatory synaptic transmission in the central nervous system of vertebrates. Expression profiles of 8 AMPAR genes were studied by RT-PCR analyses to elucidate the properties of AMPARs during early zebrafish development. Transcripts of all AMPAR genes are detected at the time of fertilization, suggesting maternal transcriptions of zebrafish AMPAR genes. The amounts of gria1 and gria2 transcripts are several-fold higher than that of gria3 and gria4 between 10 and 72 hpf (hour postfertilization). The edited gria2α transcript decreases during gastrulation period, suggesting that zygotic expression of gria2α begins around the time of midblastula transition. Relative to the amount of β-actin, the amounts of AMPAR transcripts increase significantly after the completion of neurulation. The amounts of gria2 transcripts exceed the total amounts of the remaining AMPAR transcripts after 36 hpf, suggesting increases in the representation of low Ca 2+ permeable AMPARs during neuronal maturation. Many but not all of the known mammalian protein-protein interaction motifs are preserved in the C-terminal domains (CTD) of zebrafish AMPARs. Before 16 hpf, the embryos express predominantly the alternative splice forms encoding longer CTD. Representations of the short CTD splice forms of gria2 and gria4α increase after 24 hpf, when neurulation is nearly completed.

Analytical Biochemistry, 2008

In this study, a quantitative PCR (qPCR) method was developed to determine the A-to-I RNA editing... more In this study, a quantitative PCR (qPCR) method was developed to determine the A-to-I RNA editing frequencies at specific sites. The A-to-I RNA editing of nuclear transcripts exerts profound effects on the biological activities of gene products. RNA editing of nuclear gene transcripts have been shown to be developmentally regulated and tissue specific, and alternations of RNA editing activities have been observed under pathological conditions. Two sites of ionotropic glutamate receptor subunits, the Q/R site of zebrafish gria2alpha and the Y/C site of grik2alpha, were chosen in this study to demonstrate the applicability of the SYBR Green detection-based real-time PCR method to measure RNA editing activities during zebrafish development. The results obtained by qPCR were consistent with those obtained by the limited primer extension. However, the qPCR method has the advantages of easy handling and low cost.

PLoS ONE, 2014

Background: Adar2 deaminates selective adenosines to inosines (A-to-I RNA editing) in the double-... more Background: Adar2 deaminates selective adenosines to inosines (A-to-I RNA editing) in the double-stranded region of nuclear transcripts. Although the functions of mouse Adar2 and its biologically most important substrate gria2, encoding the GluA2 subunit of AMPA (a-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptor, have been extensively studied, the substrates and functions of zebrafish Adar2 remain elusive.