Detail Expression analysis of glycogen synthase kinase-3 in human tissues and cloning of the beta-isoform promoter Bibliografi Author: Lau, Kwok Fai ; Shaw, Pang-chui (Advisor) Topik: BIOLOGY; MOLECULAR|BIOLOGY; GENETICS Bahasa: (EN )    ISBN: 0-599-23606-X     Penerbit: CHINESE UNIVERSITY OF HONG KONG (PEOPLE'S REPUBLIC OF CHINA)     Tahun Terbit: 1999     Jenis: Theses - Dissertation Fulltext: 9924017.pdf (0.0B; 0 download) Abstract Human glycogen synthase kinase-3 (GSK-3) is a multi-substrate proline-directed kinase with two isoforms -3α and -3β. Recent findings show that GSK-3 may be involved in the formation of PHF-tau protein, a hallmark in Alzheimer disease brain. In this study, the expression levels of GSK-3 RNA and proteins in different tissues were examined. Northern analysis demonstrated that GSK-3α is encoded by a 2.6-Kb mRNA and GSK-3β by 8.3- and 2.8-Kb mRNAs. The two GSK-3β mRNA species were expressed to different extents in different tissues. Both GSK-3α and -3β mRNA were prominently expressed in testis, thymus, prostate and ovary but low in adult lung and kidney. Western blot analyses showed that the 51-KD GSK-3α protein was highly expressed in lung, ovary, kidney and testis while the 46-KD GSK-3β protein was highly expressed in lung, kidney and brain. The differential expression of GSK-3α and -3β mRNA and proteins and the lack of relationship between transcription and translation in some tissues indicate that GSK-3α and -3β are subject to different means of regulation. In order to have a better understanding of the regulation of GSK-3β at transcriptional level, a DNA fragment upstream of the GSK-3β mRNA was cloned by PromoterFinder DNA walking polymerase chain reaction using primers derived from the 5′ untranslated region of the human GSK-3β cDNA. DNA with sizes 0.3, 2 and 2.5-Kb were obtained and the sequences of the former two fragments were covered by the 2.5-Kb fragment. Putative promoter region with a number of transcription factor binding sites including Sp1, CRE, MZF-1 and CCAAT were identified within the 2- and 2.5-Kb DNA fragments. These two fragments were fused with a promoterless chloramphenicol acetyl transferase (CAT) gene and CAT was detected upon transfection into COS-7 or SHSY5Y cells. GSK-3β promoter activity in SH-SY5Y cells was 50 folds higher than that in COS-7 cells, despite the transfection efficiency in SH-SY5Y cells was 10 folds lower, indicating the presence of neuronal specific transcription factors. Three transcription start sites were located by either primer extension assay or 5′ RACE. Deletion analysis indicates that positions nt −1421 to −1363 and nt −427 to −384 may contain a repressor binding site and an activator binding site, respectively. The effect of lithium, an inhibitor of GSK-3, on the promoter was also tested. It was found that lithium did not affect the GSK-3β promoter activity. Opini Anda Klik untuk menuliskan opini Anda tentang koleksi ini! Lihat Sejarah Pengadaan  Konversi Metadata   Kembali

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