Study on the cellular function of uncharacterized protein C6orf35

• Main Authors: Ya-yun Wang, 王雅筠
• Other Authors: Ih-Jen Su
• Format: Others
• Language: en_US
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/91883734682466558834

碩士 === 國立成功大學 === 分子醫學研究所 === 96 === The C6orf35 gene mapped to chromosome 6q25.3 region and encoded a novel, uncharacterized protein. As our previous work identified a 2.6 Mb common deletion interval on chromosome 6q25.2-q25.3 region was related to human EBV-associated nasal natural killer/T-cell lymphoma, it suggested that C6orf35 can be the candidate gene involved in the development of this type of lymphoma. Up to now, no report has been published for the study of C6orf35 gene. Two transcripts were predicted to be resulted from alternative splicing of C6orf35 DNA sequences. Using isoform-specific primers in commercial cDNA panels, we have previously confirmed the expressions of these two transcripts are ubiquitously in many human tissues. The predicted characteris-tics using bioinformatics analyses suggested both transcripts have transmem-brane domains, a DUF1358 domain, and may locate in the cytoplasm. To illu-strate the function of C6orf35, this study aims to generated antibodies that recognize both isoforms and used for further investigation. We have generated 5 antibodies that cover both N- and C-terminals of the deduced C6orf35 pro-teins and tested the specificity and sensitivity of these antibodies in Western blot, immunoprecipitation and immunocytochemistry analyses. Using the antibodies, the expression of C6orf35 protein in human tissues and cell lines was determined. The results showed while C6orf35L protein expresses in many human tissues and cell lines, with the most abundant expression in testis and ovary, C6orf35S protein was not detected in any of the tissues examined. Results from immunohistochemistry showed that C6orf35L protein expressed in almost all cell types in human adult testis and colon tissue. To identify the potential interacting protein of C6orf35L, co-immunoprecipitation was con-ducted and heat shock protein 70 (HSP70) and C6orf35L itself were identified by using tandem mass spectrometry analyses. In addition, we found that the expression of C6orf35L mRNA and protein were reduced under hypoxia treatment thus the data suggests the HIF1-α may be involved in the regulation of C6orf35L. Results from this study not only provide the information of ef-ficacy of C6orf35 antibodies but also improve our understanding about the characteristics of C6orf35.