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Yuen-Peng Tan,
Shaobing Li,
Xiao-Juan Jiang,
Wailin Loh,
Yik Khon Foo, Chay-Boon Loh,
Qiurong Xu,
Wai-Hong Yuen,
Michael Jones,
Jianlin Fu,
Byrappa Venkatesh,
Wei-Ping Yu
[show abstract]
[hide abstract]
ABSTRACT: The clustered protocadherins are a subfamily of neuronal cell adhesion molecules that play an important role in development of the nervous systems in vertebrates. The clustered protocadherin genes exhibit complex expression patterns in the central nervous system. In this study, we have investigated the molecular mechanism underlying neuronal expression of protocadherin genes using the protocadherin gene cluster in fugu as a model. By in silico prediction, we identified multiple neuron-restrictive silencer elements (NRSEs) scattered in the fugu protocadherin cluster and demonstrated that these elements bind specifically to NRSF/REST in vitro and in vivo. By using a transgenic Xenopus approach, we show that these NRSEs regulate neuronal specificity of protocadherin promoters by suppressing their activity in non-neuronal tissues. We provide evidence that protocadherin genes that do not contain an NRSE in their 5' intergenic region are regulated by NRSEs in the regulatory region of their neighboring genes. We also show that protocadherin clusters in other vertebrates such as elephant shark, zebrafish, coelacanth, lizard, mouse and human, contain different sets of multiple NRSEs. Taken together, our data suggest that the neuronal specificity of protocadherin cluster genes in vertebrates is regulated by the NRSE-NRSF/REST system.
Nucleic Acids Research 04/2010; 38(15):4985-97. · 8.03 Impact Factor
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Chih-Fong Chou, Chay Boon Loh,
Yik Khoon Foo,
Shuo Shen,
Burtram C Fielding,
Timothy H P Tan,
Sehaam Khan,
Yue Wang,
Seng Gee Lim,
Wanjin Hong,
Yee-Joo Tan,
Jianlin Fu
[show abstract]
[hide abstract]
ABSTRACT: Angiotensin-converting enzyme 2 (ACE2), a newly identified member in the renin-angiotensin system (RAS), acts as a negative regulator of ACE. It is mainly expressed in cardiac blood vessels and the tubular epithelia of kidneys and abnormal expression has been implicated in diabetes, hypertension and heart failure. The mechanism and physiological function of this zinc metallopeptidase in mammals are not yet fully understood. Non-mammalian vertebrate models offer attractive and simple alternatives that could facilitate the exploration of ACE2 function. In this paper we report the in silico analysis of Ace2 genes from the Gallus (chicken), Xenopus (frog), Fugu and Tetraodon (pufferfish) genome assembly databases, and from the Danio (zebrafish) cDNA library. Exon ambiguities of Danio and Xenopus Ace2s were resolved by RT-PCR and 3'RACE. Analyses of the exon-intron structures, alignment, phylogeny and hydrophilicity plots, together with the conserved synteny among these vertebrates, support the orthologous relationship between mammalian and non-mammalian ACE2s. The putative promoters of Ace2 from human, Tetraodon and Xenopus tropicalis drove the expression of enhanced green fluorescent protein (EGFP) specifically in the heart tissue of transgenic Xenopus thus making it a suitable model for future functional genomic studies. Additionally, the search for conserved cis-elements resulted in the discovery of WGATAR motifs in all the putative Ace2 promoters from 7 different animals, suggesting a possible role of GATA family transcriptional factors in regulating the expression of Ace2.
Gene 09/2006; 377:46-55. · 2.34 Impact Factor
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Choong-Tat Keng,
Aihua Zhang,
Shuo Shen,
Kuo-Ming Lip,
Burtram C Fielding,
Timothy H P Tan,
Chih-Fong Chou, Chay Boon Loh,
Sifang Wang,
Jianlin Fu,
Xiaoming Yang,
Seng Gee Lim,
Wanjin Hong,
Yee-Joo Tan
[show abstract]
[hide abstract]
ABSTRACT: The spike (S) protein of the severe acute respiratory syndrome coronavirus (SARS-CoV) interacts with cellular receptors to mediate membrane fusion, allowing viral entry into host cells; hence it is recognized as the primary target of neutralizing antibodies, and therefore knowledge of antigenic determinants that can elicit neutralizing antibodies could be beneficial for the development of a protective vaccine. Here, we expressed five different fragments of S, covering the entire ectodomain (amino acids 48 to 1192), as glutathione S-transferase fusion proteins in Escherichia coli and used the purified proteins to raise antibodies in rabbits. By Western blot analysis and immunoprecipitation experiments, we showed that all the antibodies are specific and highly sensitive to both the native and denatured forms of the full-length S protein expressed in virus-infected cells and transfected cells, respectively. Indirect immunofluorescence performed on fixed but unpermeabilized cells showed that these antibodies can recognize the mature form of S on the cell surface. All the antibodies were also able to detect the maturation of the 200-kDa form of S to the 210-kDa form by pulse-chase experiments. When the antibodies were tested for their ability to inhibit SARS-CoV propagation in Vero E6 culture, it was found that the anti-SDelta10 antibody, which was targeted to amino acid residues 1029 to 1192 of S, which include heptad repeat 2, has strong neutralizing activities, suggesting that this region of S carries neutralizing epitopes and is very important for virus entry into cells.
Journal of Virology 04/2005; 79(6):3289-96. · 5.40 Impact Factor
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Chih-Fong Chou, Chay Boon Loh,
Yik Khoon Foo,
Shuo Shen,
Burtram C. Fielding,
Timothy H.P. Tan,
Sehaam Khan,
Yue Wang,
Seng Gee Lim,
Wanjin Hong,
Yee-Joo Tan,
Jianlin Fu
[show abstract]
[hide abstract]
ABSTRACT: Angiotensin-converting enzyme 2 (ACE2), a newly identified member in the renin–angiotensin system (RAS), acts as a negative regulator of ACE. It is mainly expressed in cardiac blood vessels and the tubular epithelia of kidneys and abnormal expression has been implicated in diabetes, hypertension and heart failure. The mechanism and physiological function of this zinc metallopeptidase in mammals are not yet fully understood. Non-mammalian vertebrate models offer attractive and simple alternatives that could facilitate the exploration of ACE2 function. In this paper we report the in silico analysis of Ace2 genes from the Gallus (chicken), Xenopus (frog), Fugu and Tetraodon (pufferfish) genome assembly databases, and from the Danio (zebrafish) cDNA library. Exon ambiguities of Danio and Xenopus Ace2s were resolved by RT-PCR and 3′RACE. Analyses of the exon–intron structures, alignment, phylogeny and hydrophilicity plots, together with the conserved synteny among these vertebrates, support the orthologous relationship between mammalian and non-mammalian ACE2s. The putative promoters of Ace2 from human, Tetraodon and Xenopus tropicalis drove the expression of enhanced green fluorescent protein (EGFP) specifically in the heart tissue of transgenic Xenopus thus making it a suitable model for future functional genomic studies. Additionally, the search for conserved cis-elements resulted in the discovery of WGATAR motifs in all the putative Ace2 promoters from 7 different animals, suggesting a possible role of GATA family transcriptional factors in regulating the expression of Ace2.
Gene.
