Chi-Yao Chang

Complete Genome Sequence of the Grouper Iridovirus and Comparison of Genomic Organization with Those of Other Iridoviruses

ABSTRACT The complete DNA sequence of grouper iridovirus (GIV) was determined using a whole-genome shotgun approach on virion DNA. The circular form genome was 139,793 bp in length with a 49% G+C content. It contained 120 predicted open reading frames (ORFs) with coding capacities ranging from 62 to 1,268 amino acids. A total of 21% (25 of 120) of GIV ORFs are conserved in the other five sequenced iridovirus genomes, including DNA replication, transcription, nucleotide metabolism, protein modification, viral structure, and virus-host interaction genes. The whole-genome nucleotide pairwise comparison showed that GIV virus was partially colinear with counterparts of previously sequenced ranaviruses (ATV and TFV). Besides, sequence analysis revealed that GIV possesses several unique features which are different from those of other complete sequenced iridovirus genomes: (i) GIV is the first ranavirus-like virus which has been sequenced completely and which infects fish other than amphibians, (ii) GIV is the only vertebrate iridovirus without CpG sequence methylation and lacking DNA methyltransferase, (iii) GIV contains a purine nucleoside phosphorylase gene which is not found in other iridoviruses or in any other viruses, (iv) GIV contains 17 sets of repeat sequence, with basic unit sizes ranging from 9 to 63 bp, dispersed throughout the whole genome. These distinctive features of GIV further extend our understanding of molecular events taking place between ranavirus and its hosts and the iridovirus evolution.

Expression pattern of metallothionein, MTF‐1 nuclear translocation, and its dna‐binding activity in zebrafish (Danio rerio) induced by zinc and cadmium

Metallothionein is a small (6-kDa), cysteine-rich protein expressed by a six-zinc finger protein called metal-responsive element-binding transcription factor-1 (MTF-1) in response to Zn and Cd. Our previous reports have shown the basal expression of metallothionein (mt) and MTF-I (mtf-1) genes in embryo and early larval stages of zebrafish (Danio rerio). In the present study, we investigated the mt expression in zebrafish early larvae induced by exposure to Cd and Zn (48, 72, 96, and 120 h postfertilization). Whole-mount in situ hybridization showed that Zn induced mt expression in the olfactory pit, cerebellum, ceratobranchials, liver, chloride cells, and neuromasts of the lateral line. Cadmium also induced mt expression in all the above regions except the cerebellum. Using fluorescence techniques, we have shown that Zn and Cd mediate cytoplasmic and nuclear translocation of MTF- 1-enhanced green fluorescent protein fusion protein in zebrafish liver cell line. The MTF-1 protein was produced recombinantly by inserting zebrafish mtf-1 cDNA (1.8 kb) into pET-20b(+) expression vector and expressing in Escherichia coli BL21 (DE3) pLysS host strain competent cell on induction with isopropyl-beta-D-thiogalactopyranoside. The protein was then purified by affinity chromatography on a nickel-nitrilotriacetic acid column. Electrophoretic mobility shift assay revealed binding of the recombinant MTF-1 in response to Zn and Cd at the putative metal-responsive elements (MREs) in the promoter region of the mt gene. Taken together, these results suggest that Zn and Cd are efficiently involved with mt expression induced in zebrafish embryos and with MTF-1 nuclear translocation and that this induction is achieved through the activation of MTF-1 binding at the MREs.

Differential display of grouper iridovirus-infected grouper cells by immunostaining

Grouper iridovirus (GIV) is one of the most devastating infectious pathogens of aquaculture fish. When infecting a susceptible cell line, such as GK-2, GIV causes antigenic changes in host cellular proteins. To understand the host gene expression characteristics after viral infection, we developed an immunostaining method to screen differentially expressed genes of fish cells in response to GIV infection using phage display complementary DNA libraries. In total, 66 genes were identified from grouper kidney and brain cell lines. These genes are related to replication, transcription, translation, immunity, apoptosis, structure proteins, metabolism, energy, protein modification, and homeostasis. Four dynamic antigenic patterns were observed among these immunocloned genes upon GIV infection. Microarray analysis further confirmed the transcriptional patterns of 80% of the identified genes. This immunostaining screening method provides insights into a hosts cellular protein response to viral infection on a translational basis.

Analysis of the human adrenodoxin promoter: Evidence for its activity

Adrenodoxin is an iron-sulfur protein serving as an electron transfer intermediate in the mitochondrial cytochrome P450 system. To study its transcriptional regulation we construct a human adrenodoxin genomic clone which includes 333 bp of DNA upstream of the mRNA start site. This DNA contains a TATA box and two GC boxes. When we place it in front of the CAT reporter gene and transfect it into the recipient cell lines, it directs transcription of the CAT gene. This DNA in reverse orientation does not show any transcriptional activity. This promoter element functions in three mammalian cell lines: JEG-3, COS-1, and Y-1.

Characterization of transactivation domain and developmental expression of pituitary specific transcription factor, Pit-1 of ayu (Plecoglossus altivelis).

Pit-1 is a pituitary-specific transcription factor, which regulates the expression of growth hormone, prolactin, and thyroid stimulating hormone-beta genes. We previously reported the expression of a Pit-1 gene from ayu (Plecoglossus altivelis), which is an important cultivated food fish in Taiwan and Japan. Comparison of ayu Pit-1 with that of salmon, turkey, and rodent, revealed that the Pit-1 structure is highly conserved through vertebrates, especially in POU-specific and POU-homeo domains. The variation among fish, bird, and mammal are mainly found in transactivation domain by alternative splicing and initiation. Three insertions were found. The gamma-insert in fish Pit-1 is homologous to the exon 2a of avian Pit-1, which is not found in mammals. The beta-insert of fish Pit-1 is homologous to the 28 amino acids (a.a.) and 26 a.a. insert of avian Pit-1 beta(*) and mammalian Pit-1 beta, respectively. An additional similarity was noticed between fish and bird, as both of them contain 7 a.a. insert that is not present in mammalian Pit-1. By site directed mutagenesis, we demonstrated that the beta, gamma, and the 7 a.a. inserts of ayu Pit-1 are critical for activation of zebrafish growth hormone promoter. The ayu Pit-1 protein was found to be expressed specifically in pituitary gland, and its mRNA was first detected at embryonic day 4, significantly increased at embryonic day 5, then sustained to time of hatching at day 8.

Evolution of Alu repeats surrounding the human ferredoxin gene

Ferredoxin is an iron-sulfur protein that serves as an electron carrier for the mitochondrial oxidation/reduction system. During the characterization of the human ferredoxin gene, we have identified three Alu sequences surrounding it. When these Alu sequences were compared with others, all three of them are more related to the consensus Alu than the 7SL gene, the progenitor of the Alu family. It suggests that they are members of the modern Alu family. Their sequences differ from the Alu consensus sequence by about 5%, indicating that they were inserted into the chromosome about 35 million years ago.

Development and Application of Fish Growth Hormone Gene (Abstract)

Growth hormone is a member of a family composed of prolactin, somatolactin, and placental lactogen. It is synthesized, produced, and stored by pituitary somatotroph cells. The secreted growth hormone stimulates the production of insulin-like growth factors which mediate many of its growth-promoting effects. In this study we cloned growth hormone genes from ayu (Plecoglossus altivelis), grouper (Epinephelus awoara) and zebrafish (Danio rerio). With high homology and similarity, the putative growth hormone polypeptides contain 209, 205 and 211 amino acids respectively. Two functional recombinant GHs were expressed from E coli expression system. By inserting the zebrafish GH gene promoter region to pEGFP-1 vector, we analyzed the promoter activity by introducing the constructs into a rat pituitary cell line and zebrafish embryos. Through the fluorescent microscope, green fluorescent protein expression in the developmental stages of transgenic embryos was observed. To identify the timing and spacing of growth hormone expression during embryogenesis, whole mount in situ hybridization was also performed. As a result, growth hormone mRNA was detected as early as 1-cell and 4-cell stages. In pharyngula period, growth hormone was detected in the otic capsule and forehead region. The result of RT-PCR also showed that growth hormone expression was detected in the mature ovary, somite-stage embryos and hatched larvae. For making growth faster transgenic fish, we transferred a plasmid DNA containing carp b-actin promoter and rainbow rout growth hormone cDNA into farmed ayu by sperm-electroporation method. After five-month culture, the existence of transferred DNA was detected by PCR with specific primers in extracted genomic DNA from caudal fin tissue. The mosaic expressions of transgene were detected from gill, caudal fin, liver, brain and muscle. The growth promotion performance was indicated by 2 fold of body weight and 1.3 fold of body length.

Characterization of Alu repeats surrounding the human ferredoxin-encoding gene

Three Alu sequences have been identified surrounding the human ferredoxin-encoding genes. Among them, one is located about 1000 bp upstream from the active gene, whereas two others flank the ferredoxin pseudogene, psi FDX3. All these Alu sequences contain poly(A) tails and are flanked by direct repeats, indicating that they arose by RNA-mediated transposition events.