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Novel Transmembrane GTPase of Non-Small Cell Lung Cancer Identified by mRNA Differential Display¹

Departments of Microbiology [J-G. C.] and Biochemistry [S-L. W., Y-W. Y., H-C. H.] of the School of Medicine, Institute of Associated Chinese and Western Medicine [G-W. C.], China Medical College, Taichung 40421; Division of Chest Surgery, Department of Surgery, China Medical College Hospital [N-Y. H.], Taichung 40421; and Department of Pathology, Changhua Christian Hospital [K-T. Y.], Changhua 500, Taiwan, Republic of China

The technique of differential display was used previously to profile the gene expression patterns of non-small cell lung cancer, and several genes differentially expressed were thus identified. In this report, we demonstrate that a DNA fragment of 347-bp length, up-regulated in tumor tissues, showed 100% sequence similarity to human cDNA FLJ20693 for a 370-residue protein. The gene product of cDNA FLJ20693 was postulated to be a shorter isoform of transmembrane GTPase, termed TG370, based upon the results of searching for sequence homology. The nucleotide sequence alignment also indicated that the cDNA FLJ20693 and the cDNA for 741-residue human mitofusin 1 (TG741) possibly resulted from the event of alternative splicing from which a 127-bp region was retained in the latter. Analysis of the genome sequence confirmed the speculation that both cDNAs were mapped to the same chromosomal position composing of 18 exons, of which the 127-bp region of TG741 constituted exon 11. The alternative splicing in all lung cancer cell lines was also observed to occur nearly in all tissue specimens examined. The up-regulated expression of transmembrane GTPase was subsequently found in tumor tissues from at least five of seven non-small cell lung cancer patients. Also, a distinct PCR product was initially detected in cell line H520, and further sequence analysis identified the presence of the 86-bp region mapped to the genome sequence immediately followed by exon 10. To evaluate the retention of 86-bp region, it was found that, besides the predicted 486-bp product, an unexpected 332-bp product was concomitantly observed and identified as the result of exon 8 deletion. The expression and subcellular localization of the full-length TG741 and other shorter isoforms were detected by flow cytometry using three polyclonal antibodies. It was concluded that the full-length TG741 located at plasma membrane with its NH₂-terminal domain exposed extracellularly and the shorter isoforms retained at cytosol. Finally, the up-regulation of transmembrane GTPase in tumor tissues was further illustrated using immunohistochemical staining.

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