The polymorphism linked to the human insulin gene: its lack of association with either IDDM or NIDDM in Japanese

Abstract. Polymorphism of 5' portion of the human insulin gene was examined in 188 unrelated Japanese subjects (49 normal, 71 with IDDM, and 68 with NIDDM) using restriction endonuclease analysis. Restriction fragments were classified according to the insertion size: Class 1 (600 base pairs), Class 2 (1300 base pairs), and Class 3 (2000 base pairs). We found a very high frequency of Class 1 alleles (96.8%) and a low frequency of both Class 2 (0.8%) and Class 3 alleles (2.4%) and that approximately 94% of the genotypes were Class 1/Class 1 homozygote. In addition, there was no correlation of allelic or genotypic frequency with NIDDM or IDDM. We conclude that length polymorphism of the human insulin gene cannot be a useful marker for diabetes in Japanese.

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Low Frequency of 5'-Flanking Insertion of Human Insulin Gene in Japanese Non-Insulin-Dependent Diabetic Subjects

Diabetes Care ◽

10.2337/diacare.9.4.365 ◽

1986 ◽

Vol 9 (4) ◽

pp. 365-369 ◽

Cited By ~ 8

Author(s):

N. Aoyama ◽

T. Nakamura ◽

K. Doi ◽

S. Baba ◽

R. Takahashi ◽

...

Keyword(s):

Human Insulin ◽

Low Frequency ◽

Insulin Gene ◽

Human Insulin Gene ◽

Insulin Dependent ◽

Insulin Dependent Diabetic

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Low frequency of the large insertion in the human insulin gene in Japanese

Diabetes ◽

10.2337/diabetes.35.1.115 ◽

1986 ◽

Vol 35 (1) ◽

pp. 115-118 ◽

Cited By ~ 4

Author(s):

M. Haneda ◽

M. Kobayashi ◽

H. Maegawa ◽

Y. Shigeta

Keyword(s):

Human Insulin ◽

Low Frequency ◽

Insulin Gene ◽

Human Insulin Gene ◽

Large Insertion

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Low Frequency of the Large Insertion in the Human Insulin Gene in Japanese

Diabetes ◽

10.2337/diab.35.1.115 ◽

1986 ◽

Vol 35 (1) ◽

pp. 115-118 ◽

Cited By ~ 5

Author(s):

M. Haneda ◽

M. Kobayashi ◽

H. Maegawa ◽

Y. Shigeta

Keyword(s):

Human Insulin ◽

Low Frequency ◽

Insulin Gene ◽

Human Insulin Gene ◽

Large Insertion

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

Diabetes ◽

10.2337/diabetes.34.5.433 ◽

1985 ◽

Vol 34 (5) ◽

pp. 433-439 ◽

Cited By ~ 17

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

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Cloning and Expression of Recombinant Human Insulin Gene in Pichia pastoris

International Journal of Pharmaceutical Quality Assurance ◽

10.25258/ijpqa.10.1.22 ◽

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).

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