Cloning and sequence analysis of tumor-associated gene hMMTAG2 from human multiple myeloma cell line ARH-77.
In order to look for the tumor-associated genes from human multiple myeloma (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 in 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 cycle 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 GenBank: 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.
Tian
Jing-Yan
JY
Molecular Biology Research Center, Xiangya Medical College, Central South University, Changsha 410078, China.
Hu
Wei-Xin
WX
Tian
Er-Ming
EM
Shi
Yi-Wu
YW
Shen
Qun-Xi
QX
Tang
Li-Jun
LJ
Jiang
Yuan-Shan
YS
D016475:3T3 Cells; D000328:Adult; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002874:Chromosome Mapping; D002878:Chromosomes, Human, Pair 1 / Q000235:genetics; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D005091:Exons; D052138:Genes, Neoplasm / Q000235:genetics; D006801:Humans; D007438:Introns; D051379:Mice; D008969:Molecular Sequence Data; D009101:Multiple Myeloma / Q000235:genetics* / Q000473:pathology; D009363:Neoplasm Proteins / Q000235:genetics*; D017422:Sequence Analysis, DNA; D014162:Transfection; D014407:Tumor Cells, Cultured
C477244:C1orf35 protein, human; D018076:DNA, Complementary; D009363:Neoplasm Proteins