Se Chain Reaction (RTPCR)To identify the predominant isoform of the human PC mRNA in pancreatic beta cells, RT-PCR using human cDNA prepared from human islets and liver was performed. In this experiment, two sets of primers directed to various 59-UTR exons of the PC gene (GenBank NM_000920.3, NM_022172.2, order ML 240 BC011617.2) were designed and used in RT-PCR. Both primer sets consisted of the same sequence of the reverse primer (R-primer) and a different sequence of the forward primer (F-primer). The F-primer set no. 1 (59-ACCAACTGCCGTGATGCTGA-39) was designed to bind to the 59-UTR of variant 2 of human PC mRNA which 1531364 is transcribed by the proximal promoter while the F-primer set no. 2 (59-GATAGTGTCTGCCTTCTGGAGAGC-39) was designed to bind to the 59-UTR region of variant 3 of the human PC mRNA which is transcribed by the distal promoter. The R-primer (59ACACACGGATGGCAATCTCACC-39) was designed to bind to exon 1 of human PC mRNA [33]. Tissues were homogenized with a Qiashredder (Qiagen) (islets) or using a Potter lvehjem homogenizer (liver) and RNA was prepared using the RNeasy Mini kit (Qiagen). On-column DNase digestion was performed using the Qiagen RNase-Free DNase Set. cDNA was made with randomized primers with the Retroscript kit (AM1710) (Applied Biosystems). Quantitative PCR was performed on a BioRad MyIQ Real Time Detection System with SYBR Premix Ex Taq (RR041Q) (Takara). Human liver RNA was from a 51-year old male (Clontech, catalog number 636531) and a liver surgical specimen from a person (of unknown age and gender due to privacy protection) [34]. The PCR was carried out in a 20 mlreaction mixture containing 2 ml of cDNA, 1x PCR reaction ML-281 web buffer (20 mM Tris-HCl pH 8.4, 50 mM KCl), 0.2 mM of each primer, 100 mM of each dNTP, 2 mM MgCl2, and 1 unit Taq DNADistal Promoter of the Human Pyruvate CarboxylaseFigure 5. Identification of positive regulatory element(s) located between 2365 and 2240 of the human PC P2 promoter. (A) Schematic diagram of 15 bp internal deletions of 2114/239 the human PC P2 promoter. (B) Transient transfections of a series of 25 bp internal deletion constructs into the INS-1 832/13 cell line and non-beta cell HEK293T cell line were performed to identify the positive regulatory sequences inDistal Promoter of the Human Pyruvate Carboxylasethe hP2 promoter. The luciferase activity of each construct was normalized with b-galactosidase activity. The normalized reporter activity obtained from each construct is shown as a percent relative to those transfected with the wild type 2365 hP2 promoter, which was arbitrarily set at 100 . *P value ,0.05, **P value ,0.01. (C) Gel shift and supershift assays of the biotin-labeled probe of the 278 to 254 region of the hP2 promoter (2340/ 2315 hP2 probe) using an INS-1 832/13 nuclear extract. The nucleotide sequences of the wild type and mutant of the hP2 promoter 278 to 254 regions are also shown. Lane 1 probes incubated with nuclear extracts from 18334597 INS-1 832/13; lanes 2?, 10-fold or 50-fold excess wild-type unlabeled oligonucleotides were incubated with nuclear extracts and probes; lane 4, 50-fold excess amount of mutant unlabeled oligonucleotides were incubated with nuclear extracts and probes; lanes 5?, nuclear extracts were pre-incubated with anti-USF1 or anti-USF2 or both, respectively, before the probes were added to the reactions. Lanes 8?0, nuclear extracts were pre-incubated with anti-Sp1 or anti-Sp3 or both, respectively, before the probes were added to the reactions. Arrow rep.Se Chain Reaction (RTPCR)To identify the predominant isoform of the human PC mRNA in pancreatic beta cells, RT-PCR using human cDNA prepared from human islets and liver was performed. In this experiment, two sets of primers directed to various 59-UTR exons of the PC gene (GenBank NM_000920.3, NM_022172.2, BC011617.2) were designed and used in RT-PCR. Both primer sets consisted of the same sequence of the reverse primer (R-primer) and a different sequence of the forward primer (F-primer). The F-primer set no. 1 (59-ACCAACTGCCGTGATGCTGA-39) was designed to bind to the 59-UTR of variant 2 of human PC mRNA which 1531364 is transcribed by the proximal promoter while the F-primer set no. 2 (59-GATAGTGTCTGCCTTCTGGAGAGC-39) was designed to bind to the 59-UTR region of variant 3 of the human PC mRNA which is transcribed by the distal promoter. The R-primer (59ACACACGGATGGCAATCTCACC-39) was designed to bind to exon 1 of human PC mRNA [33]. Tissues were homogenized with a Qiashredder (Qiagen) (islets) or using a Potter lvehjem homogenizer (liver) and RNA was prepared using the RNeasy Mini kit (Qiagen). On-column DNase digestion was performed using the Qiagen RNase-Free DNase Set. cDNA was made with randomized primers with the Retroscript kit (AM1710) (Applied Biosystems). Quantitative PCR was performed on a BioRad MyIQ Real Time Detection System with SYBR Premix Ex Taq (RR041Q) (Takara). Human liver RNA was from a 51-year old male (Clontech, catalog number 636531) and a liver surgical specimen from a person (of unknown age and gender due to privacy protection) [34]. The PCR was carried out in a 20 mlreaction mixture containing 2 ml of cDNA, 1x PCR reaction buffer (20 mM Tris-HCl pH 8.4, 50 mM KCl), 0.2 mM of each primer, 100 mM of each dNTP, 2 mM MgCl2, and 1 unit Taq DNADistal Promoter of the Human Pyruvate CarboxylaseFigure 5. Identification of positive regulatory element(s) located between 2365 and 2240 of the human PC P2 promoter. (A) Schematic diagram of 15 bp internal deletions of 2114/239 the human PC P2 promoter. (B) Transient transfections of a series of 25 bp internal deletion constructs into the INS-1 832/13 cell line and non-beta cell HEK293T cell line were performed to identify the positive regulatory sequences inDistal Promoter of the Human Pyruvate Carboxylasethe hP2 promoter. The luciferase activity of each construct was normalized with b-galactosidase activity. The normalized reporter activity obtained from each construct is shown as a percent relative to those transfected with the wild type 2365 hP2 promoter, which was arbitrarily set at 100 . *P value ,0.05, **P value ,0.01. (C) Gel shift and supershift assays of the biotin-labeled probe of the 278 to 254 region of the hP2 promoter (2340/ 2315 hP2 probe) using an INS-1 832/13 nuclear extract. The nucleotide sequences of the wild type and mutant of the hP2 promoter 278 to 254 regions are also shown. Lane 1 probes incubated with nuclear extracts from 18334597 INS-1 832/13; lanes 2?, 10-fold or 50-fold excess wild-type unlabeled oligonucleotides were incubated with nuclear extracts and probes; lane 4, 50-fold excess amount of mutant unlabeled oligonucleotides were incubated with nuclear extracts and probes; lanes 5?, nuclear extracts were pre-incubated with anti-USF1 or anti-USF2 or both, respectively, before the probes were added to the reactions. Lanes 8?0, nuclear extracts were pre-incubated with anti-Sp1 or anti-Sp3 or both, respectively, before the probes were added to the reactions. Arrow rep.