Date of Award

8-2007

Level of Access Assigned by Author

Campus-Only Thesis

Degree Name

Master of Science (MS)

Department

Biochemistry

Advisor

Rebecca J. Van Beneden

Second Committee Member

Keith W. Hutchison

Third Committee Member

Gregory D. Mayer

Abstract

The aryl hydrocarbon receptor nuclear translocator (ARNT) is a member of the bHLH-PAS (basic helix-loop-helix-PER-ARNT-SIM) family of transcription factors. ARNT maintains a role in regulation of gene expression during neural development and in response to hypoxia and xenobiotic exposure in vertebrates. These processes are mediated via the hypoxia inducible factor (HIF) and aryl hydrocarbon receptor (AHR) signaling pathways, respectively. Vertebrates often contain multiple ARNT genes and splice variants. Generally, at least one form is ubiquitously expressed and others often exhibit tissue-specific expression. Invertebrate species typically contain a single ARNT homolog that exhibits tissue-dependent expression. A homolog of ARNT with four distinct variants, ARNTa, ARNTb, ARNTc and ARNTd, has been identified in the soft-shell clam, Mya arenaria. This investigation was undertaken as a first step in discovering functional roles played by ARNT gene variants in this marine invertebrate species. All four ARNT variants (ARNTa, ARNTb, ARNTc and ARNTd) contain intact PAS A domains, but two have truncated PAS B domains, which may influence interaction with their dimerization partners. Sequence analysis indicated that the four identified ARNT variants were phylogenetically related to other invertebrate ARNT homologs. Western blot analysis and quantitative PCR, revealed that ARNTa was predominant, and ubiquitously expressed, while ARNTb, ARNTc and ARNTd all exhibited tissue-dependent expression. Interestingly, although the M. arenaria ARNT forms were phylogenetically most closely related to other invertebrate homologs, the expression patterns were similar to vertebrate ARNT proteins. Since it has been suggested that ARNT is involved in reproductive processes in mammalian species, expression patterns of the clam ARNTs were examined in gonadal tissue during gamete production. ARNTa, but not ARNTb, ARNTc or ARNTd, expression changed during gametogenesis, suggesting a role of ARNTa in reproduction. Finally, because gonadal cancer has been documented in certain soft-shell clam populations along coastal Maine, clam ARNT was examined in gonadal tissue containing germinomas to determine if ARNT expression was correlated with the presence of tumors. ARNT expression in M. arenaria was not elevated in tumorigenic tissue relative to non-tumorigenic tissue, consistent with studies of human reproductive tissues. Investigation into the role that ARNT plays in the soft-shell clam provides valuable information regarding the evolutionarily conserved function of ARNT in invertebrate species. Differences in expression patterns suggest that clam ARNT gene variants are likely to be involved in different molecular pathways. ARNTa could potentially play a role in the process of gamete formation and ARNTb in hypoxia-induced pathways in the gill. Future investigations may uncover information on the evolutionarily conserved functions of protein variants in signaling pathways in an invertebrate species.

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