Skorupski J. Anim. Plant Sci., 25 (4) 2015 1129 BIOLOGICAL EFFECTS OF THE GROWTH HORMONE GENE POLYMORPHISM IN THE AMERICAN MINK (NEOVISON VISON SCHREB., 1777) - DOES SYNONYMOUS ALWAYS MEAN SILENT? Jakub J. Skorupski Department of Genetics and Animal Breeding, West Pomeranian University of Technology in Szczecin, Poland, Szczecin Corresponding author’s e-mail: jakub@gajanet.pl ABSTRACT Most often, genetic variation causing phenotypical changes is calculated exclusively upon the occurrence of non- synonymous variation. However, it has been increasingly more common to state that synonymous mutations and polymorphisms, as well as nucleotide variation present in introns, can have very serious biological effects. In the following work, the potential biological effect (including molecular phenotype) of nucleotide variation within the growth hormone gene in American mink (Neovison vison Schreb., 1777) was evaluated, based on multilocus genotypes of 389 individuals (wild mink from Canada, six colour types of ranch mink, mink acquired from the natural environment in Poland and Iceland), identified by direct sequencing. The focus is on the possible occurrence of changes in a splicing regulatory sequences and sequence motifs, different reading of codons and on an influence on the mRNA secondary structure. The results obtained confirm the hypothesis that synonymity of single-nucleotide variation does not always signify its neutrality. Key words: American mink, synonymous mutation, silent mutation, biological effect, growth hormone gene INTRODUCTION The primary measurement of nucleotide variation in a gene is the occurrence of non-synonymous variation (Loewe et al., 2006). However, it is more and more common nowadays to emphasize that synonymous mutation is actually capable of creating serious biological effects, as the synonymity is not always equal with being phenotypically neutral, especially in the molecular scope (Nackley et al., 2006, Parmley and Hurst, 2007). Moreover, a relevant biological effect can be linked to nucleotide alteration located in introns (Chorev and Carmel, 2012). The after-effects of the described genetic variation can be meaningful for changes in splicing regulatory sequences and sequence motifs, different reading of codons and for the influence on mRNA secondary structure (Shabalina et al., 2006, Parmley and Hurst, 2007, Hsu et al., 2010, Gingold and Pilpel, 2011).In the following work, the potential biological effect of nucleotide variation within the growth hormone gene in American mink is evaluated (Neovison vison Schreb., 1777) (mGH). The focus is primarily on the macromolecular phenotype, including gene expression heterogeneity and RNA phenotypes (Ferrada and Wagner, 2012, Wagner, 2014). MATERIALS AND METHODS The study involved 389 animals - 26 Canadian wild minks, 295 animals representing six farm colour- breed (Brown, Scanblack, Sapphire, Pearl, Black-Cross and Sapphire-Cross), 28 feral animals from north-west Poland and 40 from Iceland. Samples from farm animals came from slaughter-waste and from feral animals from Poland – from carcasses of animals killed on roads. In case of wild animals from Canada and feral animals from Iceland, authors have received isolated, ready-to-use DNA samples, made available by courtesy of Prof. H. Farid from Dalhousie University, and R. A. Stefánsson from West Iceland Centre of Natural History, respectively. The genomic DNA was isolated from muscle tissue (High Pure PCR Template Preparation Kit, Roche). The quality of extracted DNA was determined by agarose gel electrophoresis (AGE) on 1.0% w/v agarose. Standard PCR and nested-PCR were used to amplify the growth hormone gene. In order to obtain amplicons with an optimal length for sequencing two sets of internal and external primers were designed for two separate nested PCRs, and one set of primers for standard PCR (Tab. 1), based on the mGH sequence (GenBank: JX489617.2). DNA amplification was performed in a mixture with a volume of 15 ml. A ready-to-use 2xPCR mixture from A&A BIOTECHNOLOGY was used. All PCRs consisted of initial denaturation at 94°C for 5 min., 35 cycles of denaturation at 94°C for 40 s, annealing at 52°C (for the nested-PCR) or 55°C (standard PCR) for 40 s, and polynucleotide chain elongation at 72°C for 40s, and final extension at 72°C for 5 min. Products of standard PCR and nested-PCR with internal primers were separated by The Journal of Animal & Plant Sciences, 25(4): 2015, Page: 1129-1134 ISSN: 1018-7081