Molecular Cloning, Chromosomal Localization, And Expression Pattern Of Porcine Arf Gene Family

ADP-ribosylation factors(Arfs) are a family of GTP-binding proteins involved in fundamental biological processes including secretion,endocytosis,phagocytosis, cytokinesis,cell adhesion and tumour cell invasion.We report here the molecular cloning,chromosome localization and expression analysis of porcine Arf 1-6.The cDNA and genomic sequences of the six genes had been successfully cloned and analysed.Radiation hybrid mapping and real-time quantitative RT-PCR assays had been used to obtain the information of chromosome mapping and tissue transcription profiles.The main results are as follows:The cDNA sequences of porcine Arf1-6 including full CDS and some untranslated regions had been cloned and analysed.Of which,the coding regions of Arf1-3 all were 546bp,which all encoded 181 amino acids and porcine Arf4,5 both were 543bp, which both encoded 180 amino acids.The coding region of Arf6 was 528bp,which encoded 175 amino acids.The nucleotide sequence of porcine Arf1 shared 90%,88% and 88%similarity with its haman,mouse,rat counterparts respectively.However, the similarity of the deduced amino acid sequence all shared 100%similarity with its human,mouse,rat counterparts.The nucleotide sequence of Arf2 all have 89%with its human,mouse,rat counterparts respectively and the predicted amino acid of which all shared 99%similarity with its homologous in human,mouse and rat respectively. The nucleotide sequence of Arf3 have 94%,91%and 90%with its human,mouse,rat counterparts respectively and the predicted amino acid of which all shared 99% similarity with its homologous in human,mouse and rat respectively.The nucleotide sequence of Arf4 have 95%,94%and 93%with its human,mouse,rat counterparts respectively and the predicted amino acid of which shared 96%,98%and 98% similarity with its homologous in human,mouse and rat respectively.The nucleotide sequence of Arf5 have 95%,93%and 93%similarity with its human,mouse,rat counterparts respectively and the predicted amino acid of which all shared 100% similarity with its homologous in human,mouse and rat respectively.Arf6,the nucleotide sequence of which has 96%similarity with its human,mouse,rat counterparts respectively and the predicted amino acid of which all shared 100% similarity with its homologous in human,mouse and rat.By comparing the cDNA sequences with the corresponding genomic sequences,the genomic organization of porcine Arfs1-6 were depicted.We further characterized their genomic structures.The genomic region of porcine Classâ… Arf1-3 consisted of five exons and four introns;Classâ…¡Arf4,5 have six exons and five introns;classâ…¢Arf6 consisted of two exons and one intron,the coding region of which included in the one exon,with no intron in it.Arfs showed identical genomic structure within each class.Moreover,interspecies comparison revealed that the genomic structure of Arfs within each class is also completely conserved among mammalian species, highlighting the extremely conserved feature of Arf genes.Moreover,the splice pattern of each intron conformed to the GT-AG rule.With radiation hybrid mapping and electronic chromosome mapping,porcine Arfs1-6 are assigned to chromosome 14q21-q22,12p14,5p12-q11,13q21.1,18q24, 1q21-q27,respectively.The loci where porcine Arfs1-6 located are syntenic with the locations of their corresponding homologues on human chromosomes,providing additional evidence of interspecies conservation of Arf genes.Moreover,real-time quantitative RT-PCR assays in 10 porcine(Meishan) tissues including adipose,spleen,lung,kidney,liver,gut,brain,stomach,muscle,heart show that porcine Arfs1-6 is ubiquitous in all tissues examined,with the highest level in kidney and stomach and the lowest in muscle and heart.This is the first report of molecular characterization of Arf gene family in pigs.