Objective To analyze the upstream region of radiation-induced junB gene. Methods Four plasmids containing 250 bp, 590 bp, 900 bp and 1650 bp, and CAT reporter gene were constructed separately and introduced to L8704 c...Objective To analyze the upstream region of radiation-induced junB gene. Methods Four plasmids containing 250 bp, 590 bp, 900 bp and 1650 bp, and CAT reporter gene were constructed separately and introduced to L8704 cells. The cells were irradiated with 2 Gy X-rays and incubated at different intervals. Total RNA was extracted from the cells and fluctuation of the CAT mRNA level was assessed by the RNA ratio of CAT/β-actin measured by quantitative Northern blot hybridization. Results CAT mRNA expression containing 900 bp and 1560 bpjunB promoter remarkably and rapidly increased, and reached its peak 30min after 2 Gy X-my irradiation. Conclusions 590-900 bp fragments located in the upstream region of junB gene play an important role in the early process of cells against radiation.展开更多
Objective To screen the 5’ regulatory region of the aldose reductase (AR) gene for genetic variabilities causing changes in protein expression and affecting the promoter function. Methods The screenings were carri...Objective To screen the 5’ regulatory region of the aldose reductase (AR) gene for genetic variabilities causing changes in protein expression and affecting the promoter function. Methods The screenings were carried out by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). All SSCP variants were submitted for DNA sequencing and inserted into the plasmid chloromycetin acetyl transferase (CAT) enhancer vector. The constructs were used to transfect Hela cells,and CAT assays were performed to assess promoter activity. Gel mobility shift and footprinting assays were also performed to determine the interaction between the DNA and nuclear proteins. Results Two polymorphisms, C(-106)T and C(-12)G, were identified in the regulatory region in 123 Chinese control subjects and 145 patients with type 2 diabetes mellitus. The frequencies of genotypes WT/WT, WT/C(-12)G and WT/C(-106)T were not significantly different between the subjects and patients. In the patients with and without retinopathy, frequencies of WT/C(-106)T were 31.5% and 17.5% (P【0.05) respectively, and the frequencies of WT/C(-12)G were 10.5% and 2.5% (P】0.05) respectively. The total frequency of WT/C(-12)G and WT/C(-106)T in patients with retinopathy was 41.8%, significantly higher than that (20.0%) in patients without retinopathy (P【0.025). The relative transcription activities of the wild-type, the C(-12)G and the C(-106)T were 15.7%, 31.0% and 32.2%, respectively. The results of DNA-protein interaction assays showed that these variations did not change the binding site of DNA with trans-acting factors. Conclusion The polymorphisms C(-12)G and C(-106)T strongly associated with diabetic retinopathy in the Chinese population have been identified in the regulatory region of the aldose reductase gene.展开更多
基金This project was supported by Japanese Research Development Corporation (JRDC).
文摘Objective To analyze the upstream region of radiation-induced junB gene. Methods Four plasmids containing 250 bp, 590 bp, 900 bp and 1650 bp, and CAT reporter gene were constructed separately and introduced to L8704 cells. The cells were irradiated with 2 Gy X-rays and incubated at different intervals. Total RNA was extracted from the cells and fluctuation of the CAT mRNA level was assessed by the RNA ratio of CAT/β-actin measured by quantitative Northern blot hybridization. Results CAT mRNA expression containing 900 bp and 1560 bpjunB promoter remarkably and rapidly increased, and reached its peak 30min after 2 Gy X-my irradiation. Conclusions 590-900 bp fragments located in the upstream region of junB gene play an important role in the early process of cells against radiation.
基金grantsfromtheNationalNaturalScienceFoundationofChina (No 396 70 35 2 )
文摘Objective To screen the 5’ regulatory region of the aldose reductase (AR) gene for genetic variabilities causing changes in protein expression and affecting the promoter function. Methods The screenings were carried out by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP). All SSCP variants were submitted for DNA sequencing and inserted into the plasmid chloromycetin acetyl transferase (CAT) enhancer vector. The constructs were used to transfect Hela cells,and CAT assays were performed to assess promoter activity. Gel mobility shift and footprinting assays were also performed to determine the interaction between the DNA and nuclear proteins. Results Two polymorphisms, C(-106)T and C(-12)G, were identified in the regulatory region in 123 Chinese control subjects and 145 patients with type 2 diabetes mellitus. The frequencies of genotypes WT/WT, WT/C(-12)G and WT/C(-106)T were not significantly different between the subjects and patients. In the patients with and without retinopathy, frequencies of WT/C(-106)T were 31.5% and 17.5% (P【0.05) respectively, and the frequencies of WT/C(-12)G were 10.5% and 2.5% (P】0.05) respectively. The total frequency of WT/C(-12)G and WT/C(-106)T in patients with retinopathy was 41.8%, significantly higher than that (20.0%) in patients without retinopathy (P【0.025). The relative transcription activities of the wild-type, the C(-12)G and the C(-106)T were 15.7%, 31.0% and 32.2%, respectively. The results of DNA-protein interaction assays showed that these variations did not change the binding site of DNA with trans-acting factors. Conclusion The polymorphisms C(-12)G and C(-106)T strongly associated with diabetic retinopathy in the Chinese population have been identified in the regulatory region of the aldose reductase gene.
