Chongsuk Ryou

Access Type

Open Access Dissertation

Date of Award


Degree Type


Degree Name



Molecular Biology and Genetics

First Advisor

Dr. Richard E. Miller


Expression of a number of genes during cellular differentiation is controlled at the level of transcription. Cell type- and time-specific patterns of gene transcription result from the interaction between cis-acting elements and trans-acting factors. In order to understand the molecular mechanisms of gene expression at the level of transcription, the regulatory region of the glutamine synthetase (GS) gene was investigated using both functional and binding analyses. GS expression increases more than 100-fold during differentiation of 3T3-L1 cells into adipocytes. Recently, Hadden et al. (Nucleic Acid Research, 25:3930-3936, 1997) identified the regulatory regions of the mouse GS gene in the first intron and the distal 5'-flanking sequence. These two elements function cooperatively to regulate GS transcription during adipocyte differentiation. In my studies, I identified a putative 32 bp enhancer as well as a closely associated silencer in the first intron of the GS gene. This was accomplished by electrophoretic mobility shift analyses (EMSA) and functional analyses of stably transfected fusion genes composed of putative regulatory elements alone or in combination. To identify putative transcription factors, EMSA included the use of wild type and mutant oligonucleotides as well as the use of antibody directed against known transcription factors. My studies, taken together, revealed the nucleotide sequences required for transcriptional activity and for transcription factor binding to the 32 bp putative enhancer. During functional analyses of fusion genes, I identified a 146 bp intron 1 sequence with both enhancer and silencer activity. This sequence includes the 32 bp enhancer. Moreover, all of the enhancer activity in the 146 bp sequence resides in the 32 bp enhancer while the silencer activity resides in two elements downstream of the 32 bp enhancer. Thus, in concert with the distal 5' -flanking regulatory element interaction between positive- and negative-regulatory elements in the first intron appears to be required for transcriptional control of the native GS gene during adipocyte differentiation.