"(a) Minigene analysis: Illustration of the <t>pcDNA3.1-GNPAT</t> minigenes carrying the derived or ancestral alleles in GNPAT intron 11 (upper and lower left panels, respectively) and results of the RT-PCR and gel electrophoresis after transfection of HEK293T cells with the pcDNA3.1-GNPAT_A (A) and pcDNA3.1-GNPAT_G (G) minigenes (right panel). cDNAs were amplified with primers targeting the pcDNA3.1 5’UTR and 3’UTR regions (green arrows). Two major PCR products were detected (labeled 1 and 2). MW: Molecular weight. (b) Splicing patterns associated with the two minigenes based on Sanger sequencing of the amplicons shown in ( a ). cE: Cryptic exon. ( c ) Sequence details of the cryptic exon: The g.4,039,268G>A substitution increases the score for a predicted SF2/ASF binding site located in its 5’ region according to the ESEfinder 3.0 software. ( d ) In vivo analysis of GNPAT transcripts. Left panel, representative subset of the results obtained after gel electrophoresis following RT-PCR on total blood RNA extracted from wild-type (WT) and heterozygous (HT) Aubrac cattle using primers targeting GNPAT exons 11 and 12. Four observed PCR products are numbered and their structures are shown (see text for details). MW: Molecular weight. ( e ) Consequences of the splicing patterns shown in ( d ) on the primary structure of the <t>GNPAT</t> <t>protein.</t> Normal amino acids (AAs) are shown in green, novel AAs are shaded. The acyltransferase motif (AAs 162 to 167) and the peroxysomal targeting signal 1 (PTS1, AAs 678 to 680; (18)) are marked with an asterisk. "