Functional Analysis of Intergenic Region in Saccharomyces cerevisiae with Mass Spectrometry

• Main Authors: Wan-Jung Lee, 李宛蓉
• Other Authors: Chung-Hsuan Chen
• Format: Others
• Language: en_US
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/00969039219396177441

碩士 === 國立臺灣大學 === 化學研究所 === 101 === 　　An intergenic region (IGR) is a stretch of DNA sequence located between genes. Intergenic regions are non-coding sequences, which cannot be translated to produce proteins. Therefore, in the current cognition, the intergenic region is the so-called “junk DNA”. However, about 90% of human genome sequences belong to the intergenic region. We believe the intergenic region apparently has its reason to exist. The focus of this thesis is to investigate whether intergenic regions have functions or not. 　　In this thesis, we take Saccharomyces cerevisiae (yeast) as the model organism. The main strategy is to remove specific intergenic region of the wild-type yeast with molecular biological methods, and then use the mass spectrometry to analyze its proteomes. We call the yeast strain whose intergenic region is removed the IGR-deleted yeast strain. We compare the proteome of the deletion-type strain with the wild-type strain, and discover that some proteins have significantly changed on expression level. 　　In this study, at first we select the intergenic regions which have not been studied and showed no transcribed RNA in the database. We use homologous recombination method to place the designed anti-G418 gene (kanMX gene) into the position of intergenic region which we would like to remove. As a result, we not only successfully remove the intergenic region, but also screen the successfully IGR-deleted yeast strains according to drug-resistant characteristics. However, in order to avoid the remaining kanMX gene causing unnecessary misgivings in the later analysis results, we utilize the Cre-loxP system by transforming the phleomycin-resistent Cre-expressing vector (pSH-ble vector). With that procedure, we can remove kanMX gene and obtain the yeast strain whose intergenic region is completely removed. 　　At the same time, we use nano liquid chromatography- tandem mass spectrometer (nanoLC-MS/MS) to analyze the proteomes of two type strains, including wild-type and deletion-type yeast. Experimental results show that we can find significant changes of specific proteins. Furthermore, we have also successfully proved intergenic regions play an important role in the expression of proteins by using mass spectrometry. The most important issue we discuss currently is about whether the significant changes of specific proteins related to some biological pathways.