Masato Miyata

Viral DNA sequences of genes encoding the ATPase and the major capsid protein of tropical iridovirus isolates which are pathogenic to fishes in Japan, South China Sea and Southeast Asian countries

Summary. Tropical iridovirus infection causes severe epizootic resulting in mass mortalities and large economic losses in freshwater ornamental fishes cultured in Southeast Asian countries, in wild fish seedlings captured in South China Sea, and in marine fishes farmed in Japan, Singapore, and Thailand. All of tropical iridovirus-infected fishes histopathologically showed the systemic formation of inclusion body-bearing cells and necrosis of virus-infected splenocytes and hematopoietic cells. We designed primer sets for the ATPase gene and the major capsid protein (MCP) gene and sequenced the PCR products derived from 5 iridovirus isolates from sea bass in South China Sea, red sea bream in Japan, brown-spotted grouper with a grouper sleepy disease in Thailand, dwarf gourami from Malaysia and African lampeye from Sumatra Island, Indonesia. The ATPase gene and the MCP gene of these 5 viral isolates were highly homologous (> 95.8%, > 94.9% identity, respectively) and the deduced amino acid sequences of the ATPase and the MCP were also highly identical (> 98.1%, > 97.2% identity, respectively). Based on the high homology, these 5 isolates of tropical iridovirus from various fishes in geographically different regions were determined to have a single origin and to be native to Southeast Asian regions. However, these sequences were far different from those of members of the genera Ranavirus, Lymphocystivirus and Iridovirus in the Family Iridoviridae. We propose a new genus “Tropivirus” for tropical iridovirus in the Family Iridoviridae.

Head-to-head linkage of carp α- and β-globin genes

The α- and β-globin gene variants are believed to have diverged from a single ancestral globin gene, and the divergence was primed by the duplication of the ancestral globin gene. To understand the process of gene duplication, we investigated the α- and β-globin gene arrangement of a bony fish (carp). From a Southern analysis of seven previously prepared λ phage clones (λCG1-7) using radio-labelled α- or β-globin gene probes, it was found that the clones included both the α- and β-globin genes, and that they were located within a distance of 1 kb. Additionally, the linkage of two α-globin genes and two β-globin genes in the clone λCG1, 5 and 7 (e.g., α-β–α-β in λCG5) revealed an arrangement that is different from the arrangement in higher vertebrates in which the α-globin and β-globin genes generally occur at different loci. The distances between the detached α- to β-globin genes were approximately 5 to 10 kb. DNA sequencing of the adjacently linked α- and β-globin genes in λCG3 showed that they were arranged in a head-to-head orientation. PCR amplification using primers for the internal region between the carp α- and β-globin genes gave approximately 0.9-kb products from each of the clones λCG1, 3, 4, 5, 6, and 7, and from the chromosomal DNA of German mirror carp, Saku carp, Suwa carp, and Yamato carp. This demonstrates the alternative arrangement of carp α- and β-genes in the globin gene locus (i.e., 3′α5′-5′β3′–3′α5′-5′β3′ in λCG5), and the widespread distribution of head-to-head-linked α- and β-globin genes in carp. Based on the above results, we hypothesize that the duplication of the ancestral globin gene (prior to the divergence of the α and β forms) occurred in a head-to-head direction.

DNA cloning and characterization of an allograft inflammatory factor-1 homologue in red sea bream (Chrysophrys major)

We isolated allograft inflammatory factor-1 (AIF-1) homologue cDNA from lipopolysaccharide (LPS)-stimulated red sea bream leukocytes. We then determined that this cDNA encodes 444 bp in length and has an open reading frame that encodes 142 amino acids. The deduced amino acid sequence was homologous to the AIF-1s in carp and mammals (>65.5% sequence identity). The nucleotide sequence of the protein-coding region in the AIF-1 gene was determined to have six exons and five introns. The exon–intron structure resembles those of the mouse and the human. Genomic Southern blot analysis revealed the presence of a single AIF-1 gene per haploid in red sea bream. A time course evaluation indicated that the expression level of AIF-1 gene in LPS-stimulated leukocytes was static in the first hour, but gradually increased from 3 to 24 h. The similarities between AIF-1s in mammals and red sea bream suggest that the AIF-1 in red sea bream may have a similar function in activated leukocytes as AIF-1s do in mammals.

Gene expression and structure of globin genes of Carp

We cloned eight α- and nine β-globin genes in carp, with the basic structure of vertebrate globin genes, i.e., three exons interrupted with the two introns. In carp globin gene loci, the head-to-head arrangement suggests that the original directions of duplicated ancestral globin genes, prior to the α- and β-globin gene divergence may have occurred in the head-to-head direction. Carp globin genes are classified into 3 types (type I, type II, and type III) according to those nucleotide sequence identities of protein coding regions.