Cloning And Functional Study Of A New Growth Inhibitor Gene P28

In the large score cDNA sequencing, hundreds of full-length cDNAs of novel genes have been cloned in Chinese National Human Genome Center at Shanghai. Bioinformatics analyses indicated that one of the genes, which was named p28, was homologous to human candidate tumor suppressor gene ING1. The p28 was 249 aa long and shared 37% sequence identity with p33ING1b. There was a conserved PHD domain in C-terminal of p28 and a nuclear localization signal (NLS) upstream the PHD domain. Overexpression of ING1 inhibited cell growth, promoted the cell apoptosis and senescence. Because the p28 is homologous to ING1, we presumed that p28 had similar functions as ING1. We cloned the p28 gene by RT-PCR and constructed a series of expression plasmid constructs to study the functions of p28.The full-length of p28 is 1377bp long. The open reading frame is 747bp, encoding 249 aa. The predicted molecular weight of p28 is 28.5 kD. The p28 gene is located on 12pl3. By comparing cDNA sequences of p28 with the genomic DNA sequences, we found p28 gene contained 8 exons. Northern blot showed p28 expressed in many tissues and cultured cell lines, with high expression in skeletal muscle, placenta, heart and kidney and low expression in colon and peripheral blood. Indirect immunofluorescence displayed p28 was located in nucleus.To study the p28 functions further, we expressed and purified the GST-p28 fusion protein. By immunizing rabbits with GST-p28 fusion protein, we prepared anti-p28 polyclonal antibody with high sensitivity and specificity. We also established HepG2 stable transfected cell lines that highly expressed p28. Using these stable cell lines as a model, we...