and Sequence Analysis of Tumor-associated Gene hMMTAG2 from Human
Multiple Myeloma Cell Line ARH-77
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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