Characterization of the Distal Promoter of the Human Pyruvate Carboxylase Gene in Pancreatic Beta Cells

Pyruvate carboxylase (PC) is an enzyme that plays a crucial role in many biosynthetic pathways in various tissues including glucose-stimulated insulin secretion. In the present study, we identify promoter usage of the human PC gene in pancreatic beta cells. The data show that in the human, two alter...

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Main Authors: Ansaya Thonpho, Pinnara Rojvirat, Sarawut Jitrapakdee, Michael J. MacDonald
Other Authors: Mahidol University
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Published: 2018
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Online Access:https://repository.li.mahidol.ac.th/handle/123456789/31079
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spelling th-mahidol.310792018-10-19T12:34:22Z Characterization of the Distal Promoter of the Human Pyruvate Carboxylase Gene in Pancreatic Beta Cells Ansaya Thonpho Pinnara Rojvirat Sarawut Jitrapakdee Michael J. MacDonald Mahidol University University of Wisconsin School of Medicine and Public Health Mahasarakham University Agricultural and Biological Sciences Biochemistry, Genetics and Molecular Biology Medicine Pyruvate carboxylase (PC) is an enzyme that plays a crucial role in many biosynthetic pathways in various tissues including glucose-stimulated insulin secretion. In the present study, we identify promoter usage of the human PC gene in pancreatic beta cells. The data show that in the human, two alternative promoters, proximal and distal, are responsible for the production of multiple mRNA isoforms as in the rat and mouse. RT-PCR analysis performed with cDNA prepared from human liver and islets showed that the distal promoter, but not the proximal promoter, of the human PC gene is active in pancreatic beta cells. A 1108 bp fragment of the human PC distal promoter was cloned and analyzed. It contains no TATA box but possesses two CCAAT boxes, and other putative transcription factor binding sites, similar to those of the distal promoter of rat PC gene. To localize the positive regulatory region in the human PC distal promoter, 5′-truncated and the 25-bp and 15-bp internal deletion mutants of the human PC distal promoter were generated and used in transient transfections in INS-1 832/13 insulinoma and HEK293T (kidney) cell lines. The results indicated that positions -340 to -315 of the human PC distal promoter serve as (an) activator element(s) for cell-specific transcription factor, while the CCAAT box at -71/-67, a binding site for nuclear factor Y (NF-Y), as well as a GC box at -54/-39 of the human PC distal promoter act as activator sequences for basal transcription. © 2013 Thonpho et al. 2018-10-19T04:32:01Z 2018-10-19T04:32:01Z 2013-01-30 Article PLoS ONE. Vol.8, No.1 (2013) 10.1371/journal.pone.0055139 19326203 2-s2.0-84873804208 https://repository.li.mahidol.ac.th/handle/123456789/31079 Mahidol University SCOPUS https://www.scopus.com/inward/record.uri?partnerID=HzOxMe3b&scp=84873804208&origin=inward
institution Mahidol University
building Mahidol University Library
continent Asia
country Thailand
Thailand
content_provider Mahidol University Library
collection Mahidol University Institutional Repository
topic Agricultural and Biological Sciences
Biochemistry, Genetics and Molecular Biology
Medicine
spellingShingle Agricultural and Biological Sciences
Biochemistry, Genetics and Molecular Biology
Medicine
Ansaya Thonpho
Pinnara Rojvirat
Sarawut Jitrapakdee
Michael J. MacDonald
Characterization of the Distal Promoter of the Human Pyruvate Carboxylase Gene in Pancreatic Beta Cells
description Pyruvate carboxylase (PC) is an enzyme that plays a crucial role in many biosynthetic pathways in various tissues including glucose-stimulated insulin secretion. In the present study, we identify promoter usage of the human PC gene in pancreatic beta cells. The data show that in the human, two alternative promoters, proximal and distal, are responsible for the production of multiple mRNA isoforms as in the rat and mouse. RT-PCR analysis performed with cDNA prepared from human liver and islets showed that the distal promoter, but not the proximal promoter, of the human PC gene is active in pancreatic beta cells. A 1108 bp fragment of the human PC distal promoter was cloned and analyzed. It contains no TATA box but possesses two CCAAT boxes, and other putative transcription factor binding sites, similar to those of the distal promoter of rat PC gene. To localize the positive regulatory region in the human PC distal promoter, 5′-truncated and the 25-bp and 15-bp internal deletion mutants of the human PC distal promoter were generated and used in transient transfections in INS-1 832/13 insulinoma and HEK293T (kidney) cell lines. The results indicated that positions -340 to -315 of the human PC distal promoter serve as (an) activator element(s) for cell-specific transcription factor, while the CCAAT box at -71/-67, a binding site for nuclear factor Y (NF-Y), as well as a GC box at -54/-39 of the human PC distal promoter act as activator sequences for basal transcription. © 2013 Thonpho et al.
author2 Mahidol University
author_facet Mahidol University
Ansaya Thonpho
Pinnara Rojvirat
Sarawut Jitrapakdee
Michael J. MacDonald
format Article
author Ansaya Thonpho
Pinnara Rojvirat
Sarawut Jitrapakdee
Michael J. MacDonald
author_sort Ansaya Thonpho
title Characterization of the Distal Promoter of the Human Pyruvate Carboxylase Gene in Pancreatic Beta Cells
title_short Characterization of the Distal Promoter of the Human Pyruvate Carboxylase Gene in Pancreatic Beta Cells
title_full Characterization of the Distal Promoter of the Human Pyruvate Carboxylase Gene in Pancreatic Beta Cells
title_fullStr Characterization of the Distal Promoter of the Human Pyruvate Carboxylase Gene in Pancreatic Beta Cells
title_full_unstemmed Characterization of the Distal Promoter of the Human Pyruvate Carboxylase Gene in Pancreatic Beta Cells
title_sort characterization of the distal promoter of the human pyruvate carboxylase gene in pancreatic beta cells
publishDate 2018
url https://repository.li.mahidol.ac.th/handle/123456789/31079
_version_ 1763497179429732352