Effect of variation in 5'-regulatory region of insulin receptor substrate-1 gene on gene expression.
OBJECTIVE; To determine the effect of a variation of CAG-rich region, which was found in the 5'-regulatory sequence of insulin receptor substrate-1(IRS-1) gene in Type 2 diabetes mellitus(T2DM) patients, on gene expression and its mechanism.


METHODS
The recombinants, pGL2.P-T3 and pGL2.P-T5, were constructed with luciferase reporter vector, pGL2 promoter. T3 and T5 were wild-type and variant alleles, respectively. The recombinants were cotransfected with pSV-beta-galactosidase control vector to Hela cells. Luciferase assay was performed to assess transcriptional activity. The electrophoresis mobility shift assay(EMSA) and DNA footprint assay were applied to determine the interaction between the DNA regulatory sequences and nuclear proteins of Hela cells.


RESULTS
The relative transcription activity of T5 was lower than that of T3 [(7.76+/-1.05)% vs (9.98+/-1.40)%, P<0.05]; EMSA showed both T3 and T5 formed a single retarded band in gel with the same mobility with nuclear proteins; T5 had 2 binding sites for transacting factors, CGCGCCCGCGGGCGGCGGC and GGGCGGCTGGTGGCGGCTG, which was the same as T3.


CONCLUSIONS
Although the variation in T5 do not alter the DNA-binding sites for Hela cell nuclear extracts, the notable decrease in gene transcription activity induced by it may be an important factor to the development T2DM in the carrier.
Huang
Jian-jun
JJ
Department of Biochemistry, School of Biological Science and Technology, Central South University, Changsha 410078, China.
Liu
Hong-bo
HB
Xu
Xia
X
Song
Hui-ping
HP
D024506:5' Flanking Region; D003924:Diabetes Mellitus, Type 2 / Q000235:genetics*; D015870:Gene Expression; D014644:Genetic Variation; D006367:HeLa Cells; D006801:Humans; D055504:Insulin Receptor Substrate Proteins / Q000235:genetics*; D012045:Regulatory Sequences, Nucleic Acid
D055504:Insulin Receptor Substrate Proteins