[Increased promoter activity of human delta-globin gene with point mutation of C-T at site-64].
With PCR point mutation technique, the CCAAC box in the promoter region of delta-globin gene was changed into CCAAT which is required in the normal transcription of beta-globin gene at the corresponding site. The normal and mutant segments of the delta-globin promoter region obtained with each segment of about 300 bp (-280/+54 bp), a fragment sufficient for the requirement of its normal promoter function, were separately cloned into pUC19/Hinc II. After sequencing, it showed that the CCAAC box had been successfully changed into CCAAT without other mutations. The two segments were released by Nco I and BamH I, and were cloned into the corresponding site of plasmid pMG3 which has the firefly luciferase as its reporter gene. The positive clones were selected by Dot Blot and were comfirmed with restriction mapping. Then the two recombinant plasmids were transfected into HeLa cell line respectively and the promoter activities of the two fragments were identified by comparing the light intensity generated by the cell extracts when added with luciferase assay substrate. The results demonstrated that the luminescence of the mutant group is on average five times higher than that of the normal group, which indicate that the C-T mutation of the CCAAC box of delta-globin promoter region could increase its promoter activity. Our results have confirmed the hypothesis that the sequence of CCAAC box of human delta-globin gene is one of the main reasons which accounts for its low expression level. The author is now studying the specific DNA binding protein of the upstream promoter region of human delta-globin gene.
Zhang
J
J
Institute of Basic Medical Sciences, CAMS, Beijing.
Kou
H
H
Di
X
X
Chen
S
S
Liang
Z
Z
D005914:Globins / Q000235:genetics*; D006367:HeLa Cells; D006801:Humans; D017354:Point Mutation; D011401:Promoter Regions, Genetic / Q000235:genetics*; D014162:Transfection
D005914:Globins