Functional analysis of the GPAT4 gene mutation predicted to affect splicing.

The GPAT4 gene is considered as a potential functional candidate for single nucleotide polymorphism (SNP) studies in dairy cattle breeding due to its association with dairy performance in cattle by encoding an enzyme responsible for the presence of diacylglycerols and triacylglycerols in milk. Using the example of the GPAT4 gene, we applied the minigene splicing assay to analyze the functional consequences of its variant that was predicted to affect normal splicing. The results of functional analysis revealed the sequence variations (rs442541537), transfection experiments in a wild type and mutant cell line model system demonstrated that the investigated mutation in the second intron of the GPAT4 gene was responsible for the presence of a second exon in mature messenger RNA (mRNA). The cases of its absence in the spliced mature mRNA transcript resulted in a truncated dysfunctional protein due to the appearance of a stop codon. Thus, the discovered SNP led to alternative splicing in pre-mRNA by the 'cassette exon' ('exon skipping') mechanism. The studied mutation can potentially be a molecular genetic marker for alternative splicing for the GPAT4 gene and, therefore contributes to economic benefits in cattle breeding programs.

18S ribosomal DNA-based PCR test for avian and mammalian DNA identification in meat products.

This work has aimed to create a PCR test to identify avian and mammalian DNA in meat products. The test is based on phylogenetic analysis of 18S ribosomal RNA (rRNA) of four major groups of Tetrapod: Amphibia, Reptilia, Mammalia, and Aves. 18S rDNA complete coding sequences from GenBank have been used for phylogenetic analysis by the Maximum Likelihood method. The alignment of these 18S rDNA sequences has been used for PCR primers modeling. We have received the following PCR fragment for these primers: for birds - 97 base pairs (bp), and for mammals - 134 bp. The difference between them in 37 bp is sufficient for separating these fragments in standard agarose gel. We have tested this PCR to identify avian or mammalian DNA in sausage products and confirmed the suitability of this test for avian (chicken) and mammalian (sheep, cows) meat identification and meat identification in sausage products.