Comparative genomics identifies new alpha class genes within the avian glutathione S-transferase gene cluster Ji Eun Kim a , Miranda M. Bauer b , Kristelle M. Mendoza b , Kent M. Reed b , Roger A. Coulombe Jr. a, ⁎ a Department of Veterinary Sciences and Graduate Toxicology Program, Utah State University, Logan, UT 84322, USA b Department of Veterinary and Biomedical Sciences, University of Minnesota, St. Paul, MN 55108, USA abstract article info Article history: Received 15 September 2009 Received in revised form 2 November 2009 Accepted 3 November 2009 Available online 10 November 2009 Received by L. Marino-Ramirez Key words: Aflatoxin B1 Glutathione S-transferases Detoxification BAC Mapping SNP Glutathione S-transferases (GSTs: EC2.5.1.18) are a superfamily of multifunctional dimeric enzymes that catalyze the conjugation of glutathione (GSH) to electrophilic chemicals. In most animals and in humans, GSTs are the principal enzymes responsible for detoxifying the mycotoxin aflatoxin B 1 (AFB 1 ) and GST dysfunction is a known risk factor for susceptibility towards AFB 1 . Turkeys are one of the most susceptible animals known to AFB 1 , which is a common contaminant of poultry feeds. The extreme susceptibility of turkeys is associated with hepatic GSTs unable to detoxify the highly reactive and electrophilic metabolite exo-AFB 1 -8,9-epoxide (AFBO). In this study, comparative genomic approaches were used to amplify and identify the α-class tGST genes (tGSTA1.1, tGSTA1.2, tGSTA1.3, tGSTA2, tGSTA3 and tGSTA4) from turkey liver. The conserved GST domains and four α-class signature motifs in turkey GSTs (with the exception of tGSTA1.1 which lacked one motif) confirm the presence of hepatic α-class GSTs in the turkey. Four signature motifs and conserved residues found in α-class tGSTs are (1) xMExxxWLLAAAGVE, (2) YGKDxKERAxIDMYVxG, (3) PVxEKVLKxHGxxxL and (4) PxIKKFLXPGSxxKPxxx. A BAC clone containing the α-class GST gene cluster was isolated and sequenced. The turkey α-class GTS genes genetically map to chromosome MGA2 with synteny between turkey and human α-class GSTs and flanking genes. This study identifies the α-class tGST gene cluster and genetic markers (SNPs, single nucleotide polymorphisms) that can be used to further examine AFB 1 susceptibility and resistance in turkeys. Functional characterization of heterologously expressed proteins from these genes is currently underway. © 2009 Elsevier B.V. All rights reserved. 1. Introduction Glutathione S-transferases (GSTs; E.C.2.5.1.18), a superfamily of multifunctional dimeric proteins, are important phase II biotransfor- mation enzymes involved in cellular detoxification and excretion of a variety of xenobiotic substances (Eaton and Bammler, 1999; Frova, 2006). Carcinogens, environmental toxins and products of oxidative stress are detoxified by GSTs which principally catalyze the conjugation of reactive, electrophilic atoms with reduced glutathione (GSH) (Konishi et al., 2005; Salinas and Wong, 1999). Because of their importance in disease resistance, cancer susceptibility, and respon- siveness to drug therapy, mammalian GSTs have been intensively studied. GSTs are primarily cytosolic enzymes, but microsomal forms also exist (Kelner et al., 1996). Cytosolic GSTs exist as dimeric subunits of 23–30 k Da with an average length of 199–244 amino acids (Hayes and Pulford, 1995; Mannervik and Danielson, 1988). Each subunit is composed of two spatially distinct domains. The N-terminal domain I has an α/β structure consisting of four β-strands and three α-helices. Domain II contains a larger α domain with five to six α-helices. There are two ligand-binding sites per subunit: a specific GSH-binding site (G-site) and the hydrophobic substrate binding site (H-site) (Frova, 2006; Sun et al., 1998). Cytosolic GSTs from human, rat, and mouse have been well studied and are assigned to one of seven classes [alpha (α), mu (μ), pi (π), theta (τ), sigma (σ), zeta (ζ), omega (ο)] based on amino acid similarities (Frova, 2006; Hayes et al., 2005). Human GSTs are diverse and most abundantly expressed in the liver. Members of each class tend to have high sequence identity (N 60%)(Board, 1998) and individual genes for each human GST class are clustered together on the same chromosome (Board and Webb, 1987). Human α-class GSTs (hGSTA) are well documented with five functional genes (hGSTA1- hGSTA5) and seven pseudogenes on chromosome 6p12.1-6p12.2 (Coles and Kadlubar, 2005; Morel et al., 2002). Avian GSTs comprise a complex isoenzyme system that has received much less attention (Yeung and Gidari, 1980). According to electrophoretic mobility on SDS/PAGE, five groups of GST subunits (designated CL1–CL5) have been identified in the cytosolic fraction of Leghorn chick livers (Chang et al., 1990). Searches of Expressed Sequence Tag (EST) databases have isolated α (Chang et al., 1990; Chang et al., 1992; Liu et al., 1993), μ (Liu and Tam, 1991; Sun et al., Gene 452 (2010) 45–53 Abbreviations: GST, glutathione S-transferase; AFB1, Aflatoxin B 1 ; AFBO, exo-AFB 1 - 8,9-epoxide; tGST, turkey glutathione S-transferase. ⁎ Corresponding author. Department of Veterinary Sciences, Utah State University, Logan, UT 84322, USA. Tel.: +1 435 7971598; Fax: +1 435 797 1601. E-mail address: roger@usu.edu (R.A. Coulombe). 0378-1119/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.gene.2009.11.001 Contents lists available at ScienceDirect Gene journal homepage: www.elsevier.com/locate/gene