| Summary: | 碩士 === 中國文化大學 === 生物科技研究所 === 99 === Abstract
SPOP is a speckle-type POZ protein which is expressed ubiquitously in human tissues. The amino acid sequences of the SPOP proteins in different species are highly conserved and are 100% identical between human, mouse and rat indicating that SPOP has essential biological functions. SPOP is a TD- and POZ-domain containing protein and a member of the TD/POZ protein family. SPOP participates in protein ubiqitination and degradation. This study aimed to analyze the sequences of Spop gene promoter between species, the expression levels of Spop and the methylation status of Spop gene promoter that may correlate with the expression of Spop.
Based on NCBI information, we analyzed (1) the distribution of transcription start sites (TSS’s) and (2) alignment of the Spop promoter sequences between in different species. The 5’ RACE (rapid amplification of cDNA ends) method was used to get complete Spop cDNA 5’-end sequences, and the transcription start sites were analyzed. Four TSS’s were obtained, the results were consistent with the data from the NCBI database. According to the information from on NCBI EST database, the expression levels of the SPOP gene are different in different tissues in the mouse. Real-time PCR was used to quantify the expression levels of Spop mRNA in different tissues and cell lines in the mouse. The results showed that the expression levels of Spop mRNA were varied in different tissues and cell lines, consistent the data from the NCBI database.
To address the mechanisms of different expression, the MSP (Methylation- Specific PCR) and BSP (Bisulfite Sequencing PCR) were used to analyze the methylation status of the Spop promoter. The results showed that the CpG islands were unmethylated in the Spop promoter region in mouse tissues. The results indicate that differential expression levels of Spop between tissues and cell lines may not be due to the methylation status of the Spop promoter region. The mechanism that regulates the Spop gene expression needs to be further studied.
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