Hot topic: Gene duplication at the alpha-lactalbumin locus: finding the evidence in water buffalo (Bubalus bubalus L.).

Studies on milk proteins revealed that a qualitative and quantitative polymorphism may often be found regarding alpha-lactalbumin (alpha-LA). In mammals, a similar phenomenon was widely documented in the alpha-globin system as the result of a gene duplication. The presence of several differently expressed alpha-lactalbumin gene (LALBA) products suggests that the mechanism underlying this phenomenon may involve nonallelic genes. To check this hypothesis, an experiment was set up to investigate the LALBA gene arrangement of a water buffalo exhibiting an alpha-LA phenotype characterized by a double-band pattern on PAGE isoelectric, focusing analysis of milk protein. In particular, the relative amount of protein inferred from the different intensity of the bands was consistent with a gene duplication. Thus, leukocyte DNA was extracted from a blood sample of the buffalo and amplified with 4 primers (2 RV-IVFW for PCR and 4 FW-IRV for nested PCR). The intergenic segments of the assumed duplicated gene were then amplified with 2 different PCR protocols. First, the segment limited by the third exon in the upstream gene and the second exon in the downstream gene was amplified by simple PCR, which gave aspecific results. Second, this PCR product was subjected to nested PCR, amplifying the segment limited by the fourth exon in the upstream gene and the first exon in the downstream gene, yielding an amplified nucleotide fragment of about 6,200 bp. Blood samples from an additional 15 buffalos were then analyzed in the same manner. The results obtained from the new samples confirmed the presence of an amplified nucleotide fragment of about 6,200 bp in most of them, though they all were characterized by an alpha-LA monomorphic phenotype. A couple of 6,200-bp fragments obtained were purified, cloned in pGEM-T easy vector system (Promega, Madison, WI) and sequenced. The sequence of the large DNA segments, containing the intergenic portion, was aligned with the LALBA gene (accession number AF194373; http://www.ncbi.nlm.nih.gov/Database/index.html). They both were found to coincide with the portion containing exon 4 and the untranslated region at the 3' end of the upstream gene and with the portion containing exon 1 and the untranslated region at the 5' end of the downstream gene. These results confirm the hypothesis that a tandemly repeated copy of the LALBA gene is present in water buffalo.