Norihito Nishiyama

Characterization of Oncorhynchus mykiss 5-hydroxyisourate hydrolase/transthyretin superfamily: Evolutionary and functional analyses

Teleosts have highly diverged genomes that resulted from whole genome duplication, which leads to an extensive diversity of paralogous genes. Transthyretin (TTR), an extracellular thyroid hormone (TH) binding protein, is thought to have evolved from an ancestral 5-hydroxyisourate hydrolase (HIUHase) by gene duplication at some stage of chordate evolution. To characterize the functions of proteins that arose from duplicated genes in teleosts, we investigated the phylogenetic relationship of teleost HIUHase and TTR aa sequences, the expression levels of Oncorhynchus mykiss HIUHase and TTR mRNA in various tissues and the biological activities of the O. mykiss re-HIUHase and re-TTR. Phylogenetic analysis of the teleost aa sequences revealed the presence of two HIUHase subfamilies, HIUHase 1 (which has an N-terminal peroxisomal targeting signal-2 [PTS2]) and HIUHase 2 (which does not have an N-terminal PTS2), and one TTR family. The tissue distributions of HIUHase 1 and TTR mRNA were similar in juvenile O. mykiss and the mRNA levels were highest in the liver. The O. mykiss re-HIUHase and re-TTR proteins were both 40-50 kDa homotetramers consisting of 14-15 kDa subunits, with 30% identity. HIUHase had 5-hydroxyisourate (5-HIU) hydrolysis activity with Zn(2+) sensitivity, whereas TTR had ligand binding activity with a preference for THs and several environmental chemicals, such as halogenated phenols. Our results suggest that O. mykiss HIUHase and TTR have diverged from a common ancestral HIHUase with no functional complementation.

Identification of thyroid hormone-responsive genes in a chicken hepatoma cell line, LMH.

Abstract Thyroid hormone receptors (TRs) are ligand-dependent transcription factors that regulate the transcription of multiple thyroid hormone (TH)-responsive genes. Our study aimed to identify TH-responsive genes in an estrogen-responsive chicken hepatoma cell line, LMH. RNA was prepared from cells treated with or without 10−8 M 3,3′,5-triiodothyronine (T3) for 24 h and was analyzed by differential display. At least six cDNAs were detected whose transcript increased in the presence of T3, and four of them were cloned. The four candidate TH-responsive genes that were identified had high similarity (>83%) to known chicken or mammalian genes, which included the ribosomal protein L7 gene; the cytoplasmic dynein heavy chain gene; the scaffold attachment factor A (SAF-A) gene, also known as heterogenous nuclear ribonucleoprotein U (hnRNP U); and a gene for an unknown protein. Real-time PCR confirmed that the transcription of the four genes was responsive to T3; their transcript levels increased from four to eleven times with the administration of T3. The amount of TRβ transcript did not change with the administration of T3. The physiological reasons for the activation of these genes and the utility of this cell line are discussed.

DNA Fluorescence in situ Hybridization in Paramecium: Telomere Localization in Macronucleus

Abstract In an attempt to develop a technique of fluorescence in situ hybridization (FISH) to detect DNA in Paramecium, we examined three different DNA probes, total genomic DNA, genomic DNA encoding C5 phagosomal membrane antigen, and telomere, prepared from P. multimicronucleatum. In accordance with the conventional method, total genomic DNA probe was denatured at 75–80°C for 2 min, and the cells were denatured at 75, 80, 85, or 90°C for 5 or 10 min. The homogeneous hybridization signal with the total genomic DNA probe was obtained at 85°C for 10 min, or at 90°C for 5 min or 10 min. This condition was applied for the smaller DNA probe, C5 (1. 3 kb, the size close to detection limits), in which the expected tiny signals throughout the macronuclear nucleoplasm was observed. However, the condition was not successful for the telomeric DNA probe. The hybridization signals of telomeric DNA were only detected when both cells and probes were denatured simultaneously in a same denaturation buffer. In the case of the simultaneously denatured samples, the preservation of the nuclear morphology was relatively poor, however, the signals of the telomeric DNA probe were observed in the periphery of the macronucleus. As a negative control, an irrelevant 40 kb human cosmid probe was examined by both conventional and simultaneous denaturation methods, and none of the hybridization signal was observed with this probe. These results suggest that the current methods allow us to follow localization of the specific sequences within the macronuclear compartment.