Cloning and characterization of 5'-untranslated region of porcine beta casein gene (CSN2).
beta-Casein (CSN2) is a major milk protein in most mammals. The CSN2 gene is generally induced by lactogenic hormones bound to its promoter. The expression of this gene can be enhanced by signal transducers and activators of transcription (STAT) and glucocorticoid receptor (GR). Here, we analyzed the promoter and intron 1 regions of the porcine CSN2 gene. The porcine CSN2 promoter and intron 1 regions (-3098bp to +2446bp) were cloned into the pGL3-Basic vector containing the luciferase reporter gene (pCSN2-PEI). Lactogenic signals induced the transcription of porcine CSN2. By using AG490, a Janus kinase (JAK) inhibitor, we demonstrated that STAT5 positively regulates the transcription of porcine CSN2. Further, seven STAT mutants were generated by site-directed mutagenesis. By performing electrophoretic mobility shift assays (EMSAs), we located a critical element for pCSN2-PEI transcription bound to STAT5 in the -102bp to -84bp region. The construct containing only the promoter region (pCSN2-P), however, did not exert any promotive effects on transcription in two cell types-a mouse mammary epithelial cell line (HC11) and porcine mammary gland epithelial cells (PMECs). Thus, the construct containing intron 1 of porcine CSN2 exerts an elevating effect on transcription. We suggest that the transcription of porcine CSN2 is regulated by lactogenic signals via the STAT5 site (-102bp to -84bp) and intron 1.
Lee
Poongyeon
P
Animal Biotechnology Division, National Institute of Animal Science, RDA, Suwon, Republic of Korea.
Chung
Hee Kyoung
HK
Lee
Hyun-Gi
HG
Lee
Hwi-Cheul
HC
Woo
Jae-Seok
JS
Lee
Seunghoon
S
Jo
Su-Jin
SJ
Chang
Won-Kyong
WK
Lee
Hoon-Taek
HT
Kwon
Moosik
M
Park
Jin-Ki
JK
D020121:5' Untranslated Regions; D000818:Animals; D002364:Caseins / Q000096:biosynthesis / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D024202:Electrophoretic Mobility Shift Assay / Q000662:veterinary; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005260:Female; D005786:Gene Expression Regulation; D007438:Introns; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D050799:STAT5 Transcription Factor / Q000235:genetics; D013552:Swine / Q000235:genetics*; D014158:Transcription, Genetic / Q000502:physiology; D014162:Transfection / Q000662:veterinary; D020032:Tyrphostins / Q000494:pharmacology
D020121:5' Untranslated Regions; D002364:Caseins; D004791:Enzyme Inhibitors; D012333:RNA, Messenger; D050799:STAT5 Transcription Factor; D020032:Tyrphostins; C095512:alpha-cyano-(3,4-dihydroxy)-N-benzylcinnamide