scholarly journals The helix-loop-helix transcription factor USF (upstream stimulating factor) binds to a regulatory sequence of the human insulin gene enhancer

1993 ◽  
Vol 295 (1) ◽  
pp. 233-237 ◽  
Author(s):  
M L Read ◽  
A R Clark ◽  
K Docherty
Keyword(s):  
Transcription Factor ◽  
Human Insulin ◽  
Insulin Gene ◽  
Regulatory Sequence ◽  
Mobility Shift ◽  
Helix Loop Helix ◽  
Cell Extracts ◽  
Human Insulin Gene ◽  
Gene Enhancer ◽  
Stimulating Factor

Two important sequence elements, designated insulin enhancer binding site 1 (IEB1) or NIR and IEB2 or FAR, are involved in regulating expression of the rat insulin I gene. These elements bind a helix-loop-helix transcription factor, insulin enhancer factor 1 (IEF1). The IEB1 site is highly conserved among insulin genes but the IEB2 site is not conserved. To investigate the factors binding at the equivalent IEB1 and IEB2 sites in the human insulin gene enhancer, electrophoretic mobility shift assays were performed using a variety of cell extracts and probes specific for the homologous IEB1 and IEB2 sites. The results indicate that a factor with similar tissue distribution and binding characteristics to those of IEF1 binds to the IEB1 site in the human insulin gene, but that a separate factor, identified as the adenovirus major late transcription factor [MLTF, or upstream stimulating factor (USF)] binds to the IEB2 site.

scholarly journals Involvement of PDX-1 in activation of human insulin gene transcription

2006 ◽  
Vol 188 (2) ◽  
pp. 287-294 ◽  
Author(s):  
John Le Lay ◽  
Roland Stein
Keyword(s):  
Cell Lines ◽  
Human Insulin ◽  
Interaction Analysis ◽  
Regulatory Elements ◽  
Insulin Gene ◽  
Β Cell ◽  
Mobility Shift ◽  
Human Insulin Gene ◽  
Synergistic Activation ◽  
Gel Mobility Shift

Islet β cell-specific transcription of the insulin gene is mediated through the binding of the islet-enriched PDX-1, BETA2, and MafA transcription factors to conserved 5′-flanking region regulatory elements. However, additional non-conserved sequences within this region are also significant in regulating expression. Thus, PDX-1 binds to and activates the GG2 element located between nucleotides −145 and −140 of the human gene, while the corresponding, but non-identical, site in the rodent insulin genes are negatively regulated by the Nkx2.2 transcription factor. Here, we show that despite binding PDX-1 approximately 20-fold less effectively than the conserved insulin A3 and A1 sites in gel mobility shift assays, human GG2 appears to be more important for the activation of transfected human insulin enhancer-driven reporter constructs in β cell lines. Furthermore, functional interaction analysis in non-islet cell lines demonstrated that PDX-1 binding to GG2, A1, and A3 contributes to synergistic activation of insulin gene expression with MafA. Our analysis also illustrated the requirement of poorly conserved human sequences between −293 and −251 in mediating activity through the more upstream A3 binding site. Collectively these experiments have revealed distinct features in control of the human and rodent insulin genes by PDX-1, processes that may be involved in regulating insulin expression under both normal and diabetic conditions in humans.

scholarly journals Human insulin gene enhancer-binding proteins in pancreatic α and β cell lines

FEBS Letters ◽  
1993 ◽  
Vol 329 (1-2) ◽  
pp. 139-143 ◽  
Author(s):  
Andrew R. Clark ◽  
Helle V. Petersen ◽  
Martin L. Read ◽  
Valerie Scott ◽  
Birgitte K. Michelsen ◽  
...  
Keyword(s):  
Cell Lines ◽  
Human Insulin ◽  
Binding Proteins ◽  
Insulin Gene ◽  
Β Cell ◽  
Enhancer Binding Proteins ◽  
Human Insulin Gene ◽  
Gene Enhancer

scholarly journals Identification of a Novel PDX-1 Binding Site in the Human Insulin Gene Enhancer

2004 ◽  
Vol 279 (21) ◽  
pp. 22228-22235 ◽  
Author(s):  
John Le Lay ◽  
Taka-aki Matsuoka ◽  
Eva Henderson ◽  
Roland Stein
Keyword(s):  
Binding Site ◽  
Human Insulin ◽  
Insulin Gene ◽  
Human Insulin Gene ◽  
Gene Enhancer

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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