Structure and promoter analysis of the mouse membrane-bound transferrin-like protein (MTf) gene

Recently, we purified membrane-bound transferrin-like protein (MTf) from th e plasma membrane of rabbit chondrocytes and showed that the expression lev els of MTf protein and mRNA were much higher in cartilage than in other tis sues [Kawamoto T, Pan, H., Yan, W., Ishida, H., Usui, E., Oda, R., Nakamasu , K., Noshiro, M., Kawashima-Ohya, Y., Fujii, M., Shintani, H., Okada, Y. & Kato, Y. (1998) Eur. J. Biochem. 256, 503-509]. In this study, we isolated the MTf gene from a constructed mouse genomic library. The mouse MTf gene was encoded by a single-copy gene spanning approximate to 26 kb and consist ing of 16 exons. The transcription-initiation site was located 157 bp upstr eam from the translation-start codon, and a TATA box was not found in the 5 ' flanking region. The mouse MTf gene was mapped on the B3 band of chromoso me 16 by fluorescence in situ hybridization. Using primary chondrocytes, SK -MEL-28 (melanoma cell line), ATDC5 (chondrogenic cell line) and NIH3T3 (fi broblast cell line) cells, we carried out transient expression studies on v arious lengths of the 5' flanking region of the MTf gene fused to the lucif erase reporter gene. Luciferase activity in SK-MEL-28 cells was higher than in primary chondrocytes. Although no luciferase activity was detectable in NIH3T3 cells, it was higher in chondrocytes than in ATDC5 chondrogenic cel ls. These findings were consistent with the levels of expression of MTf mRN A in these cells cultured under similar conditions. The patterns of increas e and decrease in the luciferase activity in chondrocytes transfected with various 5' deleted constructs of the MTf promoter were similar to that in A TDC5 cells, but differed from that in SK-MEL-28 cells. The findings obtaine d with primary chondrocytes suggest that the regions between -693 and -444 and between -1635 and -1213 contain positive and negative cis-acting elemen ts, respectively. The chondrocyte-specific expression of the MTf gene could be regulated via these regulatory elements in the promoter region.