TIAN Jing-Yan, HU Wei-Xin*, TIAN Er-Ming1, SHI Yi-Wu, SHEN Qun-Xi, TANG Li-Jun, JIANG Yuan-Shan
( Molecular Biology Research Center, Xiangya Medical
College, Central South University, Changsha 410078, China;
1University of Arkansas for Medical Sciences, Arkansas Cancer
Research Center, Little Rock, AR 72205, USA )
Abstract In order to look for the tumor-associated genes from human multiple mye loma (MM), a cDNA library of human multiple myeloma cell line ARH-77 was constructed with eukaryote expression vector pcDNA3.1(+). The length of inserted fragments in library was 1.2 kb in average. All clones in cDNA library were transferred in situ to nylon membrane, which was divided into eight equal parts (A-H) and cultured in LB medium to set up gene pools. The plasmids in cDNA library and i n gene pools were extracted and NIH/3T3 cells were transfected respectively. By G418 screening and colonies counting, gene pool A was chosen for the second cycl e transfection. After several cycles, a clone, A62-17, was obtained, which had significant transforming ability. The length of this clone was 993 bp. The RACE technique was used for rapid amplification of A62-17 5'-end. The full length of this sequence has 1300 bp and was named as hMMTAG2 gene. hMMTAG2 consists of 8 exons and codes for a polypeptide of 263 amino acids (the accession number in Ge nBank: AY137773). It was located at chromosome 1q42.13. hMMTAG2 had same transforming activities in NIH/3T3 cells as the clone A62-17, and the number of transformant foci was 6 folds more than the blank vector pcDNA3.1(+). The analysis of bioinformatics revealed that hMMTAG2 had many phosphorylation sites for several protein kinases, N-myristoylation sites and nuclear localization signals, so it may be a signal molecule in the nucleus.
Key words human multiple myeloma; ARH-77 cell line; gene cloning; human multiple myeloma transforming genes (hMMTAG2)
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