| Summary: | 碩士 === 國立清華大學 === 分子與細胞生物研究所 === 93 === The major approach to study human promoters in this work is to collect promoter sequences from different primate species, and multiple sequence comparison is further performed by converting nucleotide signals to computer language through tight collaboration among biologists and computer scientists.
We have analyzed promoter sequences of some genes whose expression profiles differ significantly between human and chimpanzee brains. In order to investigate the underlying basis of nucleotides and altered gene expression, we isolated putative promoter regions of four disease-associated genes from representative New World monkeys (Saimiri sciureus), Old World monkeys (Macaca fascicularis), and great apes (Pongo pygmaeus, Gorilla gorilla, Pan troglodytes). PCR primers were designed on the basis of sequences of the first exon and the upstream region of the corresponding human gene. Our data revealed that some essential transcription factor binding sites were conserved among the primate species at the regulatory regions of these genes, suggesting that patterns of conserved sites in primate promoters may be useful to identify candidate regions crucial for gene regulation in these diseases.
Meanwhile, the sequence variation in the upstream 1 kb regions of eosinophil-derived neurotoxin (EDN, rnase2) and eosinophil cationic protein (ECP, rnase3) among human and nonhuman primates including great apes, Old World monkeys, and New World monkeys was studied. Human rnase2 and rnase3 are members of a unique subfamily of rapidly evolving primate ribonuclease genes that emerged via a gene duplication event occurring after the divergence of Old World from New World monkeys. Alignment analysis of the primate rnase2 and rnase3 promoters revealed a major difference of 34-nucleotide that appeared in promoters of all rnase2 and Old World Monkey rnase3, suggesting that a deletion of 34-nucloetide occurred in the lineage of Old World Monkey and Hominoid. In order to investigate the function of such 34-nt segment, the 1.2 kb upstream region of translation start site of human rnase2, human rnase2 with 34-nt deletion, and human rnase3 were subcloned into pGL3 reporter plasmid, and the respective transcription activity in HepG2 and K562 cells was measured. It was found that their transcription activities were similar in the K562 cells. However, interestingly, it was discovered that the 34-nucleotide contained an essential region for transactivation of rnase2 in HepG2 cells. Gel shift assay (EMSA) revealed evident mobility shift, indicating specific binding of transcription factor(s) to the promoter of human rnase2. Furthermore, the recruitment of Sp1 to the 34-nucleotide region of rnase2 in HepG2 cells was observed by DNA Affinity Precipitation Assay (DAPA). The major contribution of this work is to firstly report the deletion in the promoter region containing key transcription element(s) after gene duplication, and investigation of the correlation between such deletion and subfunctionalization.
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