Title page
ACKNOWLEDGEMENT
Contents
ABSTRACT 7
Ⅰ. INTRODUCTION 8
Ⅱ. MATERIALS AND METHODS 11
Ⅱ.1. Plasmid constructs 11
Ⅱ.2. Cell culture and transient transfection 11
Ⅲ. RESULTS 14
Ⅲ.1. Subcellular localization of hNanog wild type and deletion mutants fused to EGFP 14
Ⅲ.2. An intact homeodomain is required Ⅲ for complete hNanog nuclear localization 18
Ⅳ. DISCUSSION 22
REFERENCES 24
SUMMARY IN KOREAN 27
Table 1. Primer sequences used for cloning 13
Figure 1. Nucleocytoplasmic shuttling pathway. A protein destined for the nucleus and/or cytoplasm contains a specific sequence which can be recognized directly by importin/exportin. Nuclear import of nuclear localization signal (NLS) containing cargo can be mediated by importin / heterodimers and nuclear α β export of nuclear... 10
Figure 2. Nucleocytoplasmic localization of wild-type and deletion mutants of hNanog fused to EGFP. (A) Schematic representation of the wild-type and deletion mutants fused to EGFP. N-terminal region (NR), Homeodomain (HD), and C-terminal region (CR) are indicated. (B) Confocal laser scanning and differential... 16
Figure 3. Nucleocytoplasmic localization of EGFP-hNanog mutants containing N-terminal and C-terminal truncations of the HD fused or not the N-terminus or C-terminus of hNanog. (A) Schematic representation of the EGFP-hNanog mutants. (B) Confocal laser scanning and DIC microscopy of COS-7 cells transfected with the... 17
Figure 4. Deletion mapping of the hNanog HD. (A) Sequence alignment of the homeodomains of human Nanog and Nkx2.2 proteins. The hNanog HD shares about 45% identity with human Nkx2.2 (hNkx2.2). The hNanog NLS motifs were aligned with those of hNkx2.2. Asterisks indicate basic residues shared by hNkx2.2... 20
Figure 5. Site-directed mutagenesis of the NLS sequences within the HD. (A) Schematic representation of hNanog HD site-directed mutants fused to EGFP. (B) Confocal laser scanning and DIC microscopy of COS-7 cells transfected with EGFP-tagged fusion proteins and counter-staining of nuclear DNA with DAPI. Bar... 21