scholarly journals A tissue-specific nuclear factor binds to multiple sites in the human insulin-gene enhancer

1989 ◽  
Vol 264 (1) ◽  
pp. 233-239 ◽  
Author(s):  
D S W Boam ◽  
K Docherty
Keyword(s):  
Cell Line ◽  
Human Insulin ◽  
Specific Binding ◽  
Binding Activity ◽  
Insulin Gene ◽  
Nuclear Factor ◽  
Sequence Element ◽  
Upstream Site ◽  
Human Insulin Gene ◽  
Specific Regulation

Sequence-specific binding of proteins from an insulin-secreting cell line (RINm-5F) to the human insulin-gene 5′ region were examined by gel-retardation and methylation-interference analysis. Specific binding of a nuclear factor to sites between nucleotides -210 to -217 and -77 to -84 was detected. The same binding activity was shown at an upstream site (-313 to -320) with low affinity. Studies using mutated binding-site probes delineated a sequence 5′-C(T/C)CTAATG-3′ for high-affinity interactions. This binding activity was also present in another insulin-producing cell line (HIT.T15), but not in extracts from cell lines that did not express the insulin gene (HeLa, HL60). Cross-species comparisons show that this sequence element is highly conserved and may thus play an important role in the cell-specific regulation of insulin-gene transcription.

HEPATOCYTE NUCLEAR FACTOR-1α INHIBITS INSULIN PROMOTER FACTOR 1-DEPENDENT TRANSACTIVATION OF THE HUMAN INSULIN GENE

2001 ◽  
Vol 27 (1-2) ◽  
pp. 63-74 ◽  
Author(s):  
Kenichi Yamakawa ◽  
Hironori Yamasaki ◽  
Masako Ozaki ◽  
Mikako-Degawa Yamauchi ◽  
Naruhiro Fujita ◽  
...  
Keyword(s):  
Human Insulin ◽  
Insulin Gene ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Human Insulin Gene ◽  
Insulin Promoter ◽  
Hepatocyte Nuclear Factor 1Α

Analysis of DNA structure in the human insulin gene-linked polymorphic region in vivo

1992 ◽  
Vol 9 (3) ◽  
pp. 221-225 ◽  
Author(s):  
M. C. U. Hammond-Kosack ◽  
M. W. Kilpatrick ◽  
K. Docherty
Keyword(s):  
Cell Line ◽  
Human Insulin ◽  
Hypervariable Region ◽  
Dna Structure ◽  
Insulin Gene ◽  
Chromosomal Dna ◽  
Β Cell ◽  
Polymorphic Region ◽  
Human Insulin Gene

ABSTRACT An altered DNA structure exists within the hypervariable region located 360 bp upstream of the human insulin gene. The aim of the present study was to determine whether this structure exists in the insulin gene in vivo, and whether its presence is related to the expression of the insulin gene. However, since there were no clonal human β-cell lines available for such studies, the human insulin gene was transfected into a rat insulinoma-derived β-cell line and several human insulin-expressing clones were selected. One such cell line was treated in vivo with the DNA structural probe bromoacetaldehyde and the chromosomal DNA was extracted. Following digestion with TaqI and subsequent digestion with S1-nuclease to cleave at the bromoacetaldehydereactive sites, the DNA was subjected to agarose gel electrophoresis, and insulin gene fragments were detected by Southern blot analysis. Bromoacetaldehyde generated subfragments of 2500, 1700 and 800 bp in the human insulin gene isolated from the rat β-cell line, while the human insulin gene in the non-expressing HeLa cell line was unreactive to bromoacetaldehyde. These results suggest that an altered structure might exist in the insulin gene-linked polymorphic region of the human insulin gene in vivo, and that this structure may play a role in the expression of the insulin gene.

scholarly journals Elevated Glucose Attenuates Human Insulin Gene Promoter Activity in INS-1 Pancreatic β-Cells via Reduced Nuclear Factor Binding to the A5/Core and Z Element

2005 ◽  
Vol 19 (5) ◽  
pp. 1343-1360 ◽  
Author(s):  
Maria F. Pino ◽  
Diana Z. Ye ◽  
Katrina D. Linning ◽  
Christopher D. Green ◽  
Barton Wicksteed ◽  
...  
Keyword(s):  
Human Insulin ◽  
Promoter Activity ◽  
Gene Promoter ◽  
Insulin Gene ◽  
Nuclear Factor ◽  
Β Cells ◽  
Pancreatic Β Cells ◽  
Factor Binding ◽  
Human Insulin Gene ◽  
Elevated Glucose

Human Insulin Gene Is a Target Gene of Hepatocyte Nuclear Factor-1α (HNF-1α) and HNF-1β

1999 ◽  
Vol 263 (2) ◽  
pp. 566-569 ◽  
Author(s):  
Kohei Okita ◽  
Qin Yang ◽  
Kazuya Yamagata ◽  
Kerstin A. Hangenfeldt ◽  
Jun-ichiro Miyagawa ◽  
...  
Keyword(s):  
Human Insulin ◽  
Target Gene ◽  
Insulin Gene ◽  
Nuclear Factor ◽  
Hepatocyte Nuclear Factor ◽  
Human Insulin Gene ◽  
Hepatocyte Nuclear Factor 1Α

scholarly journals GGAAAT motifs play a major role in transcriptional activity of the human insulin gene in a pancreatic islet beta-cell line MIN6

Diabetologia ◽  
1996 ◽  
Vol 39 (12) ◽  
pp. 1462-1468 ◽  
Author(s):  
A. Tomonari ◽  
K. Yoshimoto ◽  
M. Tanaka ◽  
H. Iwahana ◽  
J. Miyazaki ◽  
...  
Keyword(s):  
Beta Cell ◽  
Cell Line ◽  
Human Insulin ◽  
Pancreatic Islet ◽  
Transcriptional Activity ◽  
Insulin Gene ◽  
Islet Beta Cell ◽  
Human Insulin Gene ◽  
Beta Cell Line ◽  
Pancreatic Islet Beta Cell

scholarly journals An insulinoma nuclear factor binding to GGGCCC motifs in human insulin gene

1993 ◽  
Vol 21 (7) ◽  
pp. 1595-1600 ◽  
Author(s):  
Louise Reibel ◽  
Corinne Besnard ◽  
Patrick Lores ◽  
Jacques Jami ◽  
Gerard Gacon
Keyword(s):  
Human Insulin ◽  
Insulin Gene ◽  
Nuclear Factor ◽  
Factor Binding ◽  
Human Insulin Gene

Lack of association of the polymorphic locus in the 5'-flanking region of the human insulin gene and diabetes in American blacks

Diabetes ◽  
1985 ◽  
Vol 34 (5) ◽  
pp. 433-439 ◽  
Author(s):  
S. Elbein ◽  
P. Rotwein ◽  
M. A. Permutt ◽  
G. I. Bell ◽  
N. Sanz ◽  
...  
Keyword(s):  
Human Insulin ◽  
Polymorphic Locus ◽  
Insulin Gene ◽  
Human Insulin Gene ◽  
Flanking Region ◽  
American Blacks

Cloning and Expression of Recombinant Human Insulin Gene in Pichia pastoris

2019 ◽  
Vol 10 (01) ◽  
Author(s):  
Rafid A. Abdulkareem
Keyword(s):  
Pichia Pastoris ◽  
Human Insulin ◽  
Expression Vector ◽  
Expression System ◽  
Insulin Gene ◽  
Cloning And Expression ◽  
Pcr Analysis ◽  
Major Band ◽  
Human Insulin Gene ◽  
Pichia Pastoris Expression System

The main goal of the current study was cloning and expression of the human insulin gene in Pichia pastoris expression system, using genetic engineering techniques and its treatment application. Total RNA was purified from fresh normal human pancreatic tissue. RNA of good quality was chosen to obtain a first single strand cDNA. Human preproinsulin gene was amplified from cDNA strand, by using two sets of specific primers contain EcoR1 and Notl restriction sites. The amplified preproinsulin gene fragment was double digested with EcoRI and Not 1 restriction enzymes, then inserted into pPIC9K expression vector. The new pPIC9K-hpi constructive expression vector was transformed by the heat-shock method into the E.coli DH5α competent cells. pPic9k –hpi, which was propagated in the positive transformant E. coli cells, was isolated from cells and then linearised by restriction enzyme SalI, then transformed into Pichia pastoris GS115 using electroporation method. Genomic DNA of His+ transformants cell was extracted and used as a template for PCR analysis. The results showed, that the pPic9k – hpi was successfully integrated into the P. pastoris genome, for selected His+ transformants clones on the anticipated band at 330 bp, which is corresponded to the theoretical molecular size of the human insulin gene. To follow the insulin expression in transformans, Tricine–SDS gel electrophoresis and Western blot analysis were conducted. The results showed a successful expression of recombinant protein was detected by the presence of a single major band with about (5.8 KDa) on the gel. These bands correspond well with the size of human insulin with the theoretical molecular weight (5.8 KDa).

scholarly journals Variation in the sequence and modification state of the human insulin gene flanking regions

1982 ◽  
Vol 10 (7) ◽  
pp. 2225-2240 ◽  
Author(s):  
Axel Ullrich ◽  
Thomas J. Dull ◽  
Alane Gray ◽  
John A. Philips ◽  
Stephan Peter
Keyword(s):  
Human Insulin ◽  
Insulin Gene ◽  
Human Insulin Gene ◽  
Flanking Regions
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