title: Cloning and characterization of the 5'-flanking region of the pig cocaine- and amphetamine-regulated transcript gene.
abstract: The cocaine- and amphetamine-regulated transcript (CART) gene encodes an anorexigenic peptide. It has a key role in the hypothalamic regulation of energy balance through reducing food intake and enhancing lipid substrate utilization. To detect the CART expression pattern in pigs, reverse transcription (RT)-polymerase chain reaction (PCR) and real-time PCR were performed in various tissues. Our RT-PCR results revealed that the pig CART gene was ubiquitously expressed in all examined tissues including hypothalamus, m. longissimus, backfat, heart, liver, spleen, lung, kidney, stomach, bladder, belly fat, brain, large intestine, lymph, and skin. Real-time quantitative PCR experiments revealed that the cDNA level of CART in both the hypothalamus and backfat of adult Landrace pig (lean-type) was significantly higher than that of Chinese indigenous Lantang pig (fat-type), and it was in the hypothalamus where the highest expression of CART was observed for both adult Lantang and Landrace pigs, compared with backfat and m. longissimus muscle. To understand the regulation of the pig CART gene, the 5'-flanking region was isolated from a pig bacterial artificial chromosome library and used in a luciferase reporter assay. A positive cis-acting element for efficient CART expression was identified at nucleotides -73 to -53, using 5'-serial deletion of the promoter. Electrophoretic mobility shift assays with competing oligonucleotides revealed that the critical region contained a cis-acting element for the zinc-binding protein factor, a zinc-finger transcription factor of the Kruppel family. This element has not been reported in human or mouse CART genes. Our results indicated that zinc-binding protein factor might be an essential regulatory factor for transcription of pig CART, providing important insight into mechanisms involved in energy homeostasis regulation in the porcine and human brain.
authors.affiliation: School of Bioscience and Bioengineering, South China University of Technology, Guangzhou, PR China.
authors.forename: Fei
authors.initials: F
authors.lastname: Ling
authors.forename: Liqiong
authors.initials: L
authors.lastname: Wei
authors.forename: Tao
authors.initials: T
authors.lastname: Wang
authors.forename: Yaosheng
authors.initials: Y
authors.lastname: Chen
authors.forename: Xiaoping
authors.initials: X
authors.lastname: Zhu
authors.forename: Jiaqi
authors.initials: J
authors.lastname: Li
authors.forename: Tingting
authors.initials: T
authors.lastname: Liu
authors.forename: Hongli
authors.initials: H
authors.lastname: Du
authors.forename: Haibo
authors.initials: H
authors.lastname: Wang
authors.forename: Jufang
authors.initials: J
authors.lastname: Wang
mesh_terms: D024506:5' Flanking Region / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation; D017930:Genes, Reporter / Q000235:genetics; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008297:Male; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics*; D009928:Organ Specificity; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion; D013552:Swine / Q000235:genetics*; D014158:Transcription, Genetic; D000098882:Cocaine- and Amphetamine-Regulated Transcript Protein
chemical_list: D009419:Nerve Tissue Proteins; D000098882:Cocaine- and Amphetamine-Regulated Transcript Protein; D008156:Luciferases