Date of Award

1996

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biological Sciences

First Advisor

Ding S. Shih

Abstract

The U3 region of long terminal repeat (LTR) of equine infectious anemia virus (EIAV) contains transcriptional regulatory elements. To identify the elements in prototype (P), fetal donkey dermal cell-adapted (A), and two variant (V) EIAV LTRs, DNase I footprinting, gel shift, and transient gene expression assays were performed. The assays used U937 monocytic cells which can be induced into macrophages by 12-O-tetradecanoylphorbol-13-acetate (TPA). For study of AP1 sites, c-Jun/AP1 was also used. Footprinting with nuclear extracts from TPA-induced and uninduced U937 cells localized methylated DNA-binding protein (MDBP), PEA2, AP1, PU.1, and CCAAT enhancer-binding protein (C/EBP) sites as common elements in 5$\sp\prime$ to 3$\sp\prime$ direction in the U3 region of the 4 LTRs. The hypervariable region between the PEA2 and AP1 sites was footprinted to contain at least one of the PEA2, AP1, or PU.1 elements. In particular, the U3 region of ALTR was footprinted to contain Oct-1, MDBP, PEA2, PEA2, AP1, PU.1, AP1, PU.1, and C/EBP sites. The Oct-1 motif exists only in ALTR. Footprinting of the 4 LTRs with purified c-Jun/AP1 and gel shift assays of oligonucleotides containing LTR AP1 sites with either c-Jun/AP1 or U937 nuclear extracts supported the identification of the AP1 sites by the footprinting with the nuclear extracts. Transient gene expression assays of the 4 wild-type LTRs and ALTR mutants were performed with TPA-induced and uninduced U937 cells. These assays identified the existence of a negative regulatory element (NRE) upstream of the MDBP site. With the exception of NRE and the PEA2 and C/EBP elements whose functions were not determined, comparison of the activities of the ALTR mutants indicated that all the elements contribute positively to both TPA-induced and uninduced basal promoter activitites. In addition, this comparison showed that the PU.1 element is the most important for the induced activity. These results suggest that the Oct-1, MDBP, AP1, and PU.1 elements cooperatively acts for the maximal basal promoter activity in monocyte and macrophage cells.

ISBN

9780591289244

Pages

158

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