Restin,a Novel Gene,Cloning And Primary Study On Its Function

The growth and development of organisms basically depend on the biological activity of cell proliferation and differentiation. As is well known that these processes is precisely modulated through cell cycle, and affected by various factors. Carcinogenesis likely occurs when these processes goes out of normal control. Great efforts have been made in studying the modulation of cell cycle, and a lot of cyclins has been found. But it remains unclear how a signal does control the cell proliferation though cell cycle. To get a deep understand in this field is to be of great importance for the control of the tumor growth and even clinical treatment of tumors. In our previous study, a series of new gene fragments was obtained by using PCR-based subtractive hybridization technique to screen cell differentiation and apoptosis-related genes from retinoic acid-treated promyelocytic cell line HL-60 cells. This work is to study the Aprl gene, one of the obtained gene fragments (GeneBank accession number AF143235), about the cloning full-length eDNA and the biological function. By the aid of computer, the Aprl cDNA is elongated to l474bp by using of EST splicing. Northern blot analysis with 12-tissue mRNA membrane reveals that the full length of this gene is about 1.5kb, which suggested that the EST "splicing sequence"is approximately the full-length. To further verify the "splicing sequence" the gene was amplified by PCR and re-sequenced. The splicing sequence has some errors; the important one is that a base pair was missed. The original sequence registered was correct and confirmed by Genebank with a new accession number of NM₀14061. Bioinformatic analysis reveals that Aprl gene encodes a 219 amino acid protein with a molecular weight about 26kD. Meanwhile, we found that Aprl protein is highly homologous (49%) to the functional domain of Necdin (neuronal growth suppressor). The two proteins are both basal proteins and belong to the MAGE (melanoma associated antigen) family. mRNA distribution analysis demonstrated that, in parallel to Necdin, this gene is mainly expressed in terminally differentiated cells. Most high expression is in nerve tissue, but very low or no expression in tumor tissues, so we renamed this gene as Restin. The melanoma antigen (MAGE) genes were initially isolated from melanomas and turned out to have an exclusively tumor-specific expression pattern. This led to the idea of using MAGE genes as targets for cancer immunotherapy, and MAGE peptides are currently being investigated as immunizing agents in clinical studies. Although 23 human and 12 mouse MAGE genes have been isolated in various tumors and characterized, not much is known about their function in normal cells. Most MAGE genes have a number of properties in common: they are pecifically expressed in a number of different tumors and in the healthy testis, they have an approximately 200-an-long MAGE-homology domain at the C-terminal end; and they are located on the X chromosome. The 23 different human MAGE genes known today have been divided into four subfamilies, A to D, based on their sequence homology and chromosomal location. The MAGE-A subfamily consists of 12 genes that are located in Xq28. The six MAGE-B genes are located in Xp21 22, and the three MAGE-C genes are found in Xq267. The two genes that constitute the MAGE-D subfamily are located in Xp 11. In adult tissues, most MAGE genes are expressed only in the testis and expression patterns suggest that this gene fam...