A Preliminary Study On The Characteristics And Functions Of MAGE-H1/Apr-1 And APMCF1 As Novel Human Genes

Upon the finish of Human genome project (HGP), revealing the function of genes is becoming a common concern of scientists all over the world. At present, the functional study on genes mainly involves the following methods: the analysis of genes' structure and characterises with bioinfomatics, their expression pattern, gene transfection in vitro, two hybrid, transgene animal, gene knock out and RNA interference which is a new technique developed recently. MAGE-H1/Apr-1 and APMCF1 were two novel human genes cloned respectively from apoptotic HL-60 and MCF-7 cells, whose functions are not clear up to now. In this research we cloned these two genes and performed some analysis and probed into their functions. 1. Preliminary study on the characteristics and functions of MAGE-H1/Apr-1At the initial stage of this research, the total RNA was isolated from the apoptotic human leukemia cell line HL-60 that was induced byall-trans-retinoic acid (ATRA). The cDNA encoding MAGE-H1/Apr-1 was amplified by reverse transcription-polymerase chain reaction (RT-PCR) method using the isolated total RNA as the template. The nucleotide sequence of the MAGE-H1/Apr-1 cDNA was found the same as in Genbank. Using genome blast, MAGE-H1/Apr-1 was mapped to chromosome Xp11.22 with the open reading frame (ORF) locating in one exon. By the aid of Bioinformatic analysis, two MAGE conserved domains were found in MAGE-H1/Apr-1. MAGE-Hl/Apr-1 shared homology with MAGE-A1, MAGE-B1, MAGE-C1, MAGE-D1 and Necdin. Phylogenetic analysis showed that MAGE-H1/Apr-1 was more closely related to MAGE-D1 and Necdin and therefore, might belong to tape II MAGE gene. To investigate the expression pattern of MAGE-H1/Apr-1 in human tissues, we analyzed the expression of MAGE-Hl/Apr-1 at messenger RNA level in human multiple tissues generated by tissue microarray (TMA). Mild expression of MAGE-H1/Apr-1 in normal human liver, adjacent nontumorous tissues and esophagus carcinoma was observed. In contrast, human primary hepatocellular carcinoma (HCC) and normal esophagus tissues were negative to MAGE-H1/Apr-1. This finding tended to suggest that MAGE-HI/Apr-1 might belong to tape II MAGE gene in terms of expression pattern. COS-7 cells transfected with pEGFP-Cl fused with MAGE-H1/Apr-1 demonstrated that MAGE-H1/Apr-1 was prominently localized at the nucleus. To test if MAGE-H1/Apr-1 played a role in the growth of liver tumor cells, we transfected MAGE-H1/Apr-1 expression constructs into HepG2 cells and examined the effect of over-expression of MAGE-H1/Apr-1 in these cells after screening a stable cell line by G418. The growth curves of the stable transfectants of tumor cells showed that HepG2 cells transfected with the MAGE-H1/Apr-1 sense gene grew much slower than the non-transfected and the control plasmid-transferred cells. Further analysis of cell cycle withflowcytometry showed that transfectants of MAGE-H1/Apr-1 could induce G1 phase arrest. This seemed to suggest that MAGE-H1/Apr-1 might play a role in the process of cell growth and that the effect of inhibiting the growth of HepG2 cells in vitro might result from the induced Gl arrest. 2. Preliminary study on the characteristics and functions of APMCF1The total RNA extracted from the apoptotic MCF-7 cells induced by ATRA was used as a template for RT-PCR in order to extend the 5' end of APMCF1. With the method of 5' rapid amplification of cDNA end (RACE) and EST assembled in GenBank, we extended the length of APMCF1 at 5' end, and as a result, a cDNA fragment with the whole length of 1745bp and containing a coding region covered 816bp was obtained at last. By using genome blast, APMCF1 was mapped to chromosome 3q22.2 and it spanned at least 14.8 kb of genomic DNA with seven exons and six introns contained. The identity of APMCF1 with the sequence compared in genome database is 100%. Using a homology search, we found that APMCF1 was homologous to the mouse signal recognition particle receptor β (SRβ) both in nucleic acid and amino acid sequence, with a 86% and 89% identity respectively. This fin...