PPO PPO, A New Putative Oncogene for Ovarian Carcinoma Therapy MD.PhD Department of orthopedic, Third Hospital HeBei Medical University Genomic Research began with The Human Genome Project (HGP), the international research effort that determined the DNA sequence of the entire human genome, completed in April Contributors to the HGP included the National Institutes of Health (NIH), which began participation in the HGP in 1988 and created the National Human Genome Research Institute (NHGRI) in 1993; the U.S. Department of Energy (DOE), where discussions of the HGP began as early as 1984; numerous universities throughout the United States; and international partners in the United Kingdom, France, Germany, Japan and China. The HGP also included efforts to characterize and sequence the entire genomes of several other organisms, many of which are used extensively in biological research. Identification of the sequence or function of genes in a model organism is an important approach to finding and elucidating the function of human genes. Human Genome Project (HGP) 1 A BAC-based STS-content map spanning a 35-Mb region of human chromosome 1p35-p36. A BAC-based STS-content map spanning a 35-Mb region of human chromosome 1p35-p36. Genomics, : 55-70 ATG TAA Genomic Structure of PPO SMART RACE (50bps) Hs (504bps) exon size intron sequences exon sequences intron sequencesintronsize gtggtcccgcggaggcggagCCCGCGGCGG AGTTTGATTGgtgcgtacgtgtggaacata ctgtactctttttttctcagGAGACAGCAAGGGGAGATCTgtgagtgaactgagaactgt ctaactttcatgtctctcagTGTGGCGCCACACGGACAGGgtaagcagagtcctcccgtg cacgatcttttcttttccagATGTGATCACACAGTGGCAGgtaggggagaaactggattt caacctgtacttgtgttcagTTTCCAGGCATCCCAGAAAGgtaaggctgcctgctcacag tttcttcttccctctcttagTGACAGCATCTGTTCAGCAGgtacaggagggtgacaggag taacgatgacttttctgtagGTTAGGGTTTCCCACTGCAGgtgagaggctgccactgctt tcctccttcttgccttccagTCTCTCGACTCTTCCCACAGgtaactgcaggcaacatggt atgccttgcttttcccccagACTGCCCTAGGAATGAAGTGgtgagtattgagaacaacag tgtccttctctttctcatagCAGAAAAGTATAGTTCAAAGgtatggtgtggcactgggca cgtgctgtctgtactcctagGACTCTCTGGGCCTGAGCGGgtaaggcagagcctttcttt gtcttgactgtggcttatagCTGTATATCCCAGCAGTTGGgtaagtagacctcattcaac aaaaatgtttcctgtagcagGGAAGGTGGTAAAAAGGCAGgtatgaacctaagaggttgg ttacttttcctttacaccagATGGCTTGCTCTCAGGCAAGgtgagtggggtttaagctcc agtatttgtttatcttgcagCTTTTTGGCAGAAGGACAAGgtgaggcatccagctctgac tgacagtttgctcattttagGAGTCGGGAATGAATACAAGgtactgtcttcagaggggat ggctctctgggtttttcaagGTCACAGCTTGCTTCGAAAGgtaggaaggcatgtcctggc cttttttgcccctgccctagGATCTCACCAGCTCTACAAGgtacatacagccagctgtct ggttttctctctctctccagAGCCTGAACCGAACTGGGTGgtggtaagcggtcactacca gccacctcctatccctgcagTACCAGTACTCACCTGCTGAgtgagtgactggggagcctc attcacttcctcctgtgtagTTGGACTACCAACAAAGCAGgtgagaccctcatgggcagt tcccggctggtgttttgcagAGAGGAGAACCACTTGTTTGgtgagtagagcccacagctg ttattccatctgccttccagGTTGTGGCCTCTCAAAGCAGgtgctttttttttttttgag agcctgggcaacagagtgagACCCTGGCTCGTAATCCCAGctacttgggaggctgtgagg aagcgaattctctgcctcagCCTCCCGAGTGGTAGACAACatcctgtgctctttcatttt Kpn I EcoR I Xba I pcDNA3.1/ V5-His 5.5 kb 1.5 kb V5 Antisense sequence Stop codon TAA-TCT XbaI site EcoRI site. Dr. Western PPO 82 KD 68 KD 55 KD 42KD V5 DAPI MERGE Vector Plasmid PPO Vector In vivo assay Days Volume PPO plasmid transfected NIH/3T3 cells vector transfected NIH/3T3 cells MEMO I PPO vector PPO MAPK vector PPO MEMO II PPO EGF EGFR P Grb2 Try1068 Sos1 Ras+ GTP membrane U0126PD98059 c-myc,c-fos c-jun expression nucleus Cytoplasm MAPK Signaling Pathway Ras+ GDP GDPs Sos PPO P P P P P activated Ras MAPK MAPKK MAPKKK MAPKKKK PI3K AKT P P CRAF ARAFBRAF MEK1/2 MAPK 1.PPO 1p36.13 2.PPO 3.PPO 4.PPO 5.PPO MAPK PPO (Proliferation and Phosphorylation Oncogene 110 KD) RT-PCR PPO/beta2 MG RT-PCR PPO HE X200 X200 HE X200 X200 X200 HE X200 X200 HE X200 X200 PPO sense RNA 5`-UAUGUUGGGAAGGUCAAGUdTdT-3` (159nt to 187nt) antisense RNA, 3`-dTdTAUACAACCCUUCCAGUUCA-5` Lamin sense RNA, 5-CUGGACUUCCAGAAGAACAdTdT-3 antisense RNA, 5-UGUUCUUCUGGAAGUCCAGdTdT-3 Vector PPO V5 p-MAPK MAPK Vector PPO beta-actin PCNA p-MEK1/2 MEK1/2 Vector PPO days Volu me A C B RNA A Lamin RNAi PPO RNAi B C PPO Lamin Volume RNA A vector PPO RNAi B C days A B PPO Lamin Volume Beta-actin PPO-RNAi 1: 2-8 2-7 PPO Knockdown 8 PPO Knockout 12 PPO PPO PPO 3 PPO sanyuanguke.org