Cloning, Tissue Expression And Imprinted Status Of Meg8 Gene In Cattle

Genomic imprinting is an epigenetic phenomenon that results in an allele-specific expression from a single in a parent-of-origin-dependent manner. Imprinted genes are genes that preferentially expressed from either the maternally allele or the paternally allele. Dlk1-Dio3 imprinted domain is conserved among mammals, and involved in embryonic, placental and muscle growth. Dlk1-Dio3 imprinted domain has been widely studied in mouse and human. But due to the lack of sequence and polymorphism information in coding regions, few imprinted genes have been reported in cattle.In this study, we cloned the cDNA sequence and analyzed the expression and imprinted status of Meg8 gene, which is identified as a maternally expressed gene in both mouse and sheep. The cDNA sequences were obtained by RT-PCR and RACE (GenBank accession number: HQ407410-HQ407422). Sequence analyse showed that 12 transcript variants occured in cattle Meg8 gene. The DNA and cDNA sequences were about 43 kb and 1062 bp, respectively. There are 7 exons in cattle Meg8 gene,the exons had high identity ranged from 82.8 to 97.7% when aligned with the sheep orthologue. Bioinformatics analysis showed that none of the ATGs is consistent with Kozak consensus sequence. So the cattle Meg8 gene may be a noncoding RNA and act as a regulated factor. These results are essential to analyze the biological function and the molecular regulation mechanism of cattle Meg8 gene.We used RT-PCR to analyse the tissue-specific distribution of the cattle Meg8 gene in different tissues.The results indicated that Meg8 was expressed in all investigated tissues, including heart, liver, spleen, lung, kidney, brain, skeletal muscle and subcutaneous fat of the adult cattle. Our data showed that no specific expression pattern existed in cattle Meg8 gene which is not consistent with mouse and sheep.Expression patterns of imprinted genes are studied based on the fact that the genes are monoallelically expressed. To study if gene expressed as monoallelic or biallelic, an expressed polymorphism (polymorphism in the mRNA or proteins) must be found to distinguish which parental allele is transcribed. In addition, the animals being studied must be heterozygous for the gene/polymorphism of interest. In the mammalian genome single nucleotide polymorphisms (SNP) are the most common polymorphisms, which are DNA point mutations (base-pair change or insertions/deletions) and can be distributed in the genome level. Single strand conformation polymorphism(SSCP) is a quite simple, rapid and reproducible method and widely used in the detection of known mutations and the analysis of DNA variation.We found an SNP in the coding sequence of cattle Meg8 gene by PCR-SSCP and then analysed the cDNA of the heterozygous individuals using RT-PCR and direct sequencing. Results revealed that the Meg8 gene was monoallelically expressed in the heart, liver, spleen, lung, kidney, brain, skeletal muscle and subcutaneous fat in adult cattle. This is the first time to determine that the Meg8 gene is imprinted in cattle.