Lamb P, Crawford L
Mol Cell Biol. 1986 May;6(5):1379-85. doi: 10.1128/mcb.6.5.1379-1385.1986.
Cosmid and lambda clones containing the human p53 gene were isolated and characterized in detail. The gene is 20 kilobases (kb) long and has 11 exons, the first and second exons being separated by an intron of 10 kb. Restriction fragments upstream of sequences known to be within the first identified exon were tested for promoter activity by cloning them in front of the chloramphenicol acetyltransferase gene and transfecting the resulting constructs into HeLa cells. A 0.35-kb DNA fragment was identified that had promoter activity. Results of primer extension experiments indicated that the mRNA cap site falls within this fragment, as expected. Analysis of the sequence upstream of the presumptive cap site indicated that the human p53 promoter may be of an unusual type.
分离并详细鉴定了含有人p53基因的黏粒和λ噬菌体克隆。该基因长20千碱基(kb),有11个外显子,第一和第二外显子被一个10 kb的内含子隔开。通过将已知位于第一个鉴定外显子内的序列上游的限制性片段克隆到氯霉素乙酰转移酶基因前,并将所得构建体转染到HeLa细胞中,来检测其启动子活性。鉴定出一个具有启动子活性的0.35 kb DNA片段。引物延伸实验结果表明,正如预期的那样,mRNA帽位点位于该片段内。对假定帽位点上游序列的分析表明,人p53启动子可能属于一种不寻常的类型。