Cloning Of Novel Genes Related Apoptosis By Improved, PCR-based Subtractive Hybridization

Apoptosis is a kind of cell death in human and other multicellular organism, it is neither always abnormal nor always detrimental, and the homeostatic balance of the number of cells is determined by both proliferation and apoptosis of cells. Deregulation of apoptosis is associated with the pathogenesis of a wide array of diseases: cancer, neurodegeneration, autoimmunity, heart disease, and other disorders. The further understanding of apoptosis always follows the discovery of a novel gene involved in this process, and inspires people to think more deeply about the molecular mechanism of a disease. So, it has profound significance to clone apoptosis related genes.In this study, the promyelocytic cell line HL-60 cells were induced to apoptosis by ATRA, the apoptotic cDNA library was constructed and 32 fragments differentially expressed in apoptotic HL-60 cells were cloned after a new version of PCR-based Subtractive hybridization. There were 5 false positive clones identified by reverse dot blot, and the rest 27 clones were identified as apoptotic related genes. These clones were sequenced, then analyzed with BLAST and ORF analysis, and 17 of 27 clones were genes already known, and the left 10 were identified as novel genes. 7 of 17 genes were reported participate apoptosis, and the function of rest 10 of the 17 genes is not clear. 5 of the 10 novel genes are sequenced thoroughly. These 5 genes were named respectively from apr-1 to apr-5, and accepted by GenBank. Their accession numbers are AF143235, AF143236, AF144055, AF144054, and AF144056. The other 5 clones have relative long fragments, the sequencing has not finished yet.