SHORT COMMUNICATION Characterization and mapping of bovine dopamine receptors 1 and 5 A. Haegeman*, J. L. Williams † , A. Law † , A. Van Zeveren* and L. J. Peelman* *Department of Animal Nutrition, Genetics, Breeding and Ethology, Faculty of Veterinary Medicine, Ghent University, Merelbeke, Belgium. † Department of Genomics and Bioinformatics, Roslin Institute (Edinburgh), Roslin, Midlothian EH25 9PS, Scotland Summary A cDNA encoding the bovine dopamine receptor 1 (DRD1) was isolated from a bovine cDNA library, cloned and completely sequenced. The coding region showed 93 and 91% sequence identity on DNA level and 96 and 94% on protein level with its respective porcine and human orthologs. The bovine DRD1 and dopamine receptor 5 (DRD5) were mapped, respectively, to BTA10 and 6 by radiation hybrid mapping. One SNP was found in DRD1 and four in DRD5. Using polymerase chain reaction–restriction fragment length poly- morphism, 11 different European cattle breeds were screened for the presence of the DRD1 and DRD5 substitutions. Allele frequencies for DRD1 and DRD5 alleles were very similar across all the breeds examined. Allele frequency discrepancies were found between Belgian Blue beef breed and the other breeds. Keywords complementary deoxyribonucleic acid, dopamine receptor, mutation, polym- erase chain reaction, restriction fragment length polymorphism, SSCP. Maintaining a balance between calorie uptake and expenditure is of vital importance for each living organism. Therefore, it is not surprising that the energy balance is a complex system, which is regulated by a great number of pathways. Chronic excessive calorific uptake or fasting will upset that balance and will result in risks to health (Kopelman 2000). Prime candidates in regulating energy homeostasis are the dopamine receptors (DRDs) of which currently five are identified. Their importance in energy homeostasis has been shown by the association of allelic variants (of DRD2 and DRD4) with risk of obesity (Comings et al. 1993; Noble et al. 1994; Blum et al. 1996; Poston et al. 1998). In addition, dopamine receptors have been implicated in the regulation of feeding behaviour (Martel & Fantino 1996; Szczypka et al. 1999; Lutz et al. 2001). Furthermore, interaction of the DRDs with other factors implicated in obesity, such as neuropeptide Y (NPY), corti- cotropin releasing hormone (CRH), have been demonstrated (Bina & Cincotta 2000; Kuo 2002). In spite of this evidence, very little is known about the dopamine receptors in cattle with the exception of DRD2, which has been completely sequenced (Chio et al. 1990) and mapped to BTA15 (Amarante et al. 1999). In order to search for potential SNPs, primer pairs were designed using the Primer 3 program (Rozen & Skaletsky 1998). Primer sequences, the annealing temperatures and the obtained amplified lengths are shown in Table 1. The identity of all the DRD1 and DRD5 polymerase chain reac- tion (PCR) fragments were determined by sequencing (ALFexpress TM. Autoread TM. Sequencing Kit; Pharmacia, Uppsala, Sweden) and comparing the sequences obtained with the National Center for Biotechnology Information (NCBI) database. Three DNA samples, from five different cattle breeds (Red Holstein, Red Pied, Red West Flanders, Belgian Blue and Blonde d’Aquitaine) were investigated for the presence of possible mutations using SSCP, as described in Haegeman et al. (2001). Altered SSCP patterns were identified in the DRD1 ± 1 and DRD5 ± 1 PCR fragments. The possible mutations were identified by sequencing and multiple sequence alignment. A single base substitution [T (allele B) to C (allele A)] was identified in the DRD1 fragment. However, four mutations were observed in the DRD5 fragment, namely three C–T and one G–A. Only the G (allele A) to A (allele B) transition results in an amino acid change (G fi S). The nucleotide substitution in the DRD1 (T fi C) and DRD5 fragment (G fi A) can be detected using PCR–restriction fragment length polymorphism (RFLP). For the DRD1 fragment, digestion with RsaI (creation of a RsaI restriction site fi GTAC) resulted in fragment lengths of 204 bp (allele B) and/or 154 + 50 bp (allele A). For bovine DRD5, a specific primer was designed (DRD5 ) 2), based upon sequence data obtained. Digestion with PstI (CTGCAG) of the DRD5 fragments resulted in either a 87-bp fragment Address for correspondence Dr Luc J. Peelman, Department of Animal Nutrition, Genetics, Breeding and Ethology, Faculty of Veterinary Medicine, Ghent University, Heidestraat 19, 9820 Merelbeke, Belgium. E-mail: luc.peelman@rug.ac.be Accepted for publication 10 February 2003 Ó 2003 International Society for Animal Genetics, Animal Genetics, 34, 290–293 290