B Chen1, Z Guo, P He, P Ye, C Buresi, G Roizes. 1. Department of Molecular Biology & Biochemistry, Institute of Basic Medical Science, Chinese Academy of Medical Science, Beijing 100005, China.
Abstract
OBJECTIVE: To study the structure of alleles in the 3' end of the apoB gene in Han, Mongolian and Tibetan populations in China as well as the roles in the regulation of gene expression. METHODS: DNA were obtained from human leukocytes by phenol-chloroform extraction and ethanol precipitation. PCR were carried out in a 50 microliters volume containing 50 ng genomic DNA as template. The Ssp1-digested products were loaded on a gradient acrylamide gel and run for 3 hours. The constructs containing alleles were tested in cultured HepG2 and HeLa cells using transient assays. RESULTS: Sixteen alleles with different repeat number were characterized. All of the alleles varying from HVE22 to HVE52, allele HVE34 was the most common (58.4%), followed by allele HVE36 (13.8%) and HVE32 (10.5%). 258 PCR products were digested with Ssp1 and run in 4-12% PAGE. We detected the fragments of 266bp, 91 bp, 61 bp and 39 bp in almost all samples. The small alleles (including HVE22, HVE24, HVE26 and HVE36) decreased the expressive activity of the luciferase reporter, in contrary, the large alleles (including HVE44, HVE46 and HVE48) elevated obviously the expressive activity of the luciferase reporter. CONCLUSIONS: More alleles with different number of tandem repeats in 3' end of apoB gene exist in the Chinese populations. The alleles in 3' end minisatellite of human apoB gene could control the expression of the gene itself.
OBJECTIVE: To study the structure of alleles in the 3' end of the apoB gene in Han, Mongolian and Tibetan populations in China as well as the roles in the regulation of gene expression. METHODS: DNA were obtained from human leukocytes by phenol-chloroform extraction and ethanol precipitation. PCR were carried out in a 50 microliters volume containing 50 ng genomic DNA as template. The Ssp1-digested products were loaded on a gradient acrylamide gel and run for 3 hours. The constructs containing alleles were tested in cultured HepG2 and HeLa cells using transient assays. RESULTS: Sixteen alleles with different repeat number were characterized. All of the alleles varying from HVE22 to HVE52, allele HVE34 was the most common (58.4%), followed by allele HVE36 (13.8%) and HVE32 (10.5%). 258 PCR products were digested with Ssp1 and run in 4-12% PAGE. We detected the fragments of 266bp, 91 bp, 61 bp and 39 bp in almost all samples. The small alleles (including HVE22, HVE24, HVE26 and HVE36) decreased the expressive activity of the luciferase reporter, in contrary, the large alleles (including HVE44, HVE46 and HVE48) elevated obviously the expressive activity of the luciferase reporter. CONCLUSIONS: More alleles with different number of tandem repeats in 3' end of apoB gene exist in the Chinese populations. The alleles in 3' end minisatellite of humanapoB gene could control the expression of the gene itself.