Isolation of the regulatory regions and genomic organization of the porcine alpha1,3-galactosyltransferase gene.

BACKGROUND Alpha1,3-galactosyltransferase (alpha1,3GT) is an enzyme that produces carbohydrate chains termed alphaGal epitopes found in most mammals, although some species of higher primates, including human, are notable exceptions. The evolutionary origin of the lost alpha1,3GT enzyme activity is not yet known, although it has been suggested that the promoter activity of this gene in the ancestors of higher primates was inactivated. METHODS We used 5'-or 3'-RACE, GenomeWalking, reverse transcriptase polymerase chain reaction (RT-PCR) and dual Luciferase reporter assay for identification of the full-length cDNA, which includes the transcription initiation site and the promoter region of porcine alpha1,3GT gene. RESULTS The region around exon 1 is guanine and cytosine (GC)-rich (about 70%), comprising a CpG island spanning more than 1.5 kbp. The 5'-flanking region of exon 1 contains multiple transcription factor consensus motifs, including GC-box, SP1, AP2, and GATA-box sites, in the absence of TATA or CAAT-box sequences. The entire gene consists of three 5' noncoding and six coding region exons spanning more than 52 kbp. Detailed analysis of alpha1,3GT transcripts revealed two major alternative splicing patterns in the 5'-untranslated region (5'-UTR) and evidence for minor splicing activity that occurs in a tissue-specific manner. Interspecies comparison of 5'-UTR shows minimal homology between porcine and murine sequences except for exon 2, which suggests that the regulatory regions differ among species. CONCLUSIONS These observations have important implications for experiments involving genetic manipulation of the alpha1,3GT gene in transgenic animals in terms of promoter utilization, and particularly in genetically engineering cells for the animal cloning technology by nuclear transfer.