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Transforming Growth Factor-ß (Tgf-ß) in Teleost Fish: Development of a Quantitative Assay for mRNA Expression and Application to a Field Study of Fish Health
EPA Grant Number: U915209Title: Transforming Growth Factor-ß (Tgf-ß) in Teleost Fish: Development of a Quantitative Assay for mRNA Expression and Application to a Field Study of Fish Health
Investigators: Harms, Craig A.
Institution: North Carolina State University
EPA Project Officer: Boddie, Georgette
Project Period: January 1, 1997 through January 1, 2000
Project Amount: $102,000
RFA: STAR Graduate Fellowships (1997)
Research Category: Fellowship - Molecular Biology/Genetics , Biology/Life Sciences , Academic Fellowships
Description:
Objective:The general hypothesis of this research project are to: (1) determine whether cytokines homologous to those characterized in mammals are present in fish; and (2) assess whether cytokine production will provide a powerful tool for monitoring the immune status in a broad range of fish species in response to toxicologic insults and infectious agents. Research is focused on transforming growth factor-ß (TGF-ß), a cytokine which plays an important role in immunity (being primarily immunosuppressive), development, cell proliferation, and carcinogenesis.
Approach:A TGF-ß was isolated and cloned from hybrid striped bass (Morone saxatilis x Morone. chrysops) anterior kidney mononuclear cells. This isolate contains an open reading frame of 1,146 bases coding for a 382 amino acid protein most similar to rainbow trout TGF-ß (57.3% percent and 78.6% percent identity with precursor and active protein respectively) and rat TGF-ß1 (41.1% percent and 68.8% percent identity with precursor and active protein respectively). Consensus primers were designed, which amplify—by polymerase chain reaction (PCR)Ca TGF-ß segment from at least 11 species of teleost fish comprising 8eight taxonomic families in 5five orders. A reverse transcription quantitative competitive polymerase chain reaction (RT-qcPCR) assay was devised to measure TGF-ß mRNA expression in teleost fish. The RT-qcPCR assay was used to determine TGF-ß transcription levels from mononuclear cells of different lymphoid tissues of hybrid striped bass. The assay was then used to compare TGF-ß mRNA expression with macrophage bacteriocidal activity in hybrid striped bass in response to an immunomodulator and in white perch (Morone americana) in a field study.
Supplemental Keywords:fellowship, teleost fish, polymerase chain reaction, PCR, reverse transcription quantitative competitive polymerase chain reaction, RT-qcPCR, cytokine production, cell proliferation.