This research concerns the hormonal regulation of the vitellogenins and the apolipoproteins of very low density lipoprotein. These proteins are synthesized in the avian liver in response to estrogenic stimulation and serve to transport lipid and other nutrients to the yolk for embryonic development. These proteins are of great interest as models for the study of estrogen action and because of their roles in lipid transport. This proposal is largely focused on the regulation of apolipoprotein II (apo II) gene expression. Apo II is a major protein component of the very low density lipoprotein particles synthesized in response to estrogen. The estrogen-stimulated synthesis of apo II involves activation of gene transcription and a marked cytoplasmic stabilization of apo II mRNA. Experiments will be carried out to examine the estrogen-mediated stabilization of apo II mRNA and to test the hypothesis that stabilization is due to site specific interactions between stabilization factors and apo II mRNA. The research will be directed to three major questions. First, how is apo II mRNA structure related to functional aspects of its expression? These studies will require elucidation of the heterogeneity found at the 5' and 3' ends of the mRNA and an analysis of apo II mRNA secondary structure. A new method for high resolution mapping of mRNAs with base and struture specific ribonucleases has been developed for this purpose. Second, What is the pattern of protein binding on the mRNA and how does it relate to secondary structure and stabiliztion of apo II mRNA? Nuclease mapping will be used to determine sequences that are altered as a result of interaction with protein. Third, do apo II messenger ribonucleoprotein particles contain proteins unique to this mRNA? Are such proteins involved in estrogen-mediated stabilization. These particles will be purified and the proteins analyzed. Monoclonal antibodies to apo II mRNP proteins will be used to determine which proteins are unique to apo II mRNA, which are common to etrogen-regulated mRNAs, and which are common to all mRNAs. The long-term goal is to understand how estrogen-mediated stabilization of mRNA occurs. This is a major action of estrogens that has received relatively little attention.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK018171-13
Application #
3225959
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1978-02-01
Project End
1990-01-31
Budget Start
1987-02-01
Budget End
1988-01-31
Support Year
13
Fiscal Year
1987
Total Cost
Indirect Cost
Name
State University New York Stony Brook
Department
Type
Schools of Medicine
DUNS #
804878247
City
Stony Brook
State
NY
Country
United States
Zip Code
11794
Margot, J B; Williams, D L (1996) Estrogen induces the assembly of a multiprotein messenger ribonucleoprotein complex on the 3'-untranslated region of chicken apolipoprotein II mRNA. J Biol Chem 271:4452-60
Sensel, M G; Binder, R; Lazier, C B et al. (1994) Reactivation of apolipoprotein II gene transcription by cycloheximide reveals two steps in the deactivation of estrogen receptor-mediated transcription. Mol Cell Biol 14:1733-42
MacDonald, C C; Williams, D L (1993) RNase H/oligonucleotide-directed mRNA purification (ROMP) of apoll mRNA. Nucleic Acids Res 21:765-6
MacDonald, C C; Williams, D L (1992) Proteins associated with the messenger ribonucleoprotein particle for the estrogen-regulated apolipoprotein II mRNA. Biochemistry 31:1742-8
Nicosia, M; Prack, M M; Williams, D L (1992) Differential regulation of apolipoprotein-E messenger RNA in zona fasciculata cells of rat adrenal gland determined by in situ hybridization. Mol Endocrinol 6:288-98
Nicosia, M; Moger, W H; Dyer, C A et al. (1992) Apolipoprotein-E messenger RNA in rat ovary is expressed in theca and interstitial cells and presumptive macrophage, but not in granulosa cells. Mol Endocrinol 6:978-88
Prack, M M; Nicosia, M; Williams, D L et al. (1991) Relationship between apolipoprotein E mRNA expression and tissue cholesterol content in rat adrenal gland. J Lipid Res 32:1611-8
Binder, R; Gordon, D A; Hwang, S P et al. (1990) Estrogen-induced destabilization and associated degradation intermediates of apolipoprotein II mRNA. Prog Clin Biol Res 322:227-40
Fenjves, E S; Lee, J I; Garlick, J A et al. (1990) Prospects for epithelial gene therapy. Basic Life Sci 53:215-23
Binder, R; MacDonald, C C; Burch, J B et al. (1990) Expression of endogenous and transfected apolipoprotein II and vitellogenin II genes in an estrogen responsive chicken liver cell line. Mol Endocrinol 4:201-8

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