Graduate Thesis Or Dissertation
 

Molecular characterization of the MX genes of rainbow trout (Oncorhynchus mykiss)

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  • The Mx protein family of rainbow trout (Oncorhynchus mykiss) was characterized at the molecular level. With primers derived from a partial sequence of Perch (Perca fluviatis) Mx genomic DNA, a partial rainbow trout Mx genomic DNA sequence was obtained by polymerase chain reaction (PCR) amplification. All salmonid fish investigated contained Mx homologous DNA sequences as determined by Southern blot. Northern blot analysis of Mx expression demonstrated that Mx mRNA was induced by poly inosinic cytidylic (IC) dsRNA both in vitro and in vivo and by infectious hematopoietic necrosis virus (IHNV) in vivo. RACE-PCR (rapid amplification of cDNA ends-polymerase chain reaction) was used to clone three Mx cDNA clones (designated RBTMx) from the rainbow trout gonad cell line, RTG-2, after induction by poly IC dsRNA. The deduced RBTMx 1, 2, and 3 proteins are 621, 636, and 623 amino acids in length with molecular weights of 70.6, 72, and 70.8 kD respectively. The trout Mx proteins share approximately 50% identity at the amino acid level with mammalian and avian Mx proteins and contain the tripartite GTP binding domain and leucine zipper motifs common to all Mx proteins. Polyclonal antisera generated to an E. coli expressed trout Mx protein fragment recognized all three trout Mx proteins. Poly IC dsRNA induced the expression of trout Mx protein in vitro. Mx protein was detected in the liver and kidney of rainbow trout challenged with IHNV. Immunohistochemical staining demonstrated that Mx protein was expressed in the kidney tubules of rainbow trout following exposure to IHNV. Trout Mx cDNA clones were transiently expressed under the control of a eukaryotic viral promoter in a salmon embryo cell line by transfection. RBTMx1 was expressed in the cytoplasm in regions surrounding the nucleus. Staining for RBTMx2 produced a punctate pattern within the nucleus, characteristic of mammalian and rodent Mx proteins, and in the cytoplasm of some cells. RBTMx3 was expressed in a diffuse pattern, uniformly distributed throughout the cytoplasm, but not within the nucleus. Transient expression of the trout Mx proteins did not result in inhibition of II-INV replication as evidenced by nucleocapsid protein accumulation.
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