The Production and Characterization of Xenopus Albumin Complementary-Dna Clones and Regulation of Rna Levels by Estrogen and Dexamethasone
Jackson, Cynthia Lou
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https://hdl.handle.net/2142/70525
Description
Title
The Production and Characterization of Xenopus Albumin Complementary-Dna Clones and Regulation of Rna Levels by Estrogen and Dexamethasone
Author(s)
Jackson, Cynthia Lou
Issue Date
1983
Department of Study
Biochemistry
Discipline
Biochemistry
Degree Granting Institution
University of Illinois at Urbana-Champaign
Degree Name
Ph.D.
Degree Level
Dissertation
Keyword(s)
Biology, General
Abstract
In order to study the regulation of Xenopus albumin by estrogen and dexamethasone, albumin cDNA clones were isolated from an 18S cDNA library produced from uninduced RNA. Several albumin clones were identified and characterized with respect to their restriction maps, and homology to each other. Clones corresponding to both 74,000 and 72,000 dalton albumin were isolated. There appears to be a minimum of three groups of clones represented.
These clones were used as hybridization probes to study the regulation of albumin mRNA in an organ culture system. Albumin mRNA gradually declined in cells maintained in the absence of hormones. Cells maintained in the presence of 2 x 10('-6) M dexamethasone and 1 x 10('-8)MT(,3) continue to produce albumin mRNA, although at a reduced level relative to in vivo synthesis. Cells withdrawn from hormones in culture, could be induced to produce albumin mRNA to a greater level than that seen with cells continuously maintained in the presence of dexamethasone and T(,3) in a dose dependent fashion. When estrogen is added to liver cultures grown in the presence of 2 x 10('-6) M dexamethasone and 1 x 10('-8)MT(,3) the amount of albumin mRNA begins to decline. Albumin mRNA begins to decline after three days of growth in estrogen and declines in a linear fashion from 3 to 10 days, declining by 80% over that time period.
In order to facilitate the quantitation of the albumin mRNA in the large number of samples generated by regulatory studies, a spot hybridization method was developed. This method was shown to be quantitative and yield similar results to solution hybridization by measuring the reinduction of vitellogenin mRNA in Xenopus, withdrawn from estrogen for an extended period of time. The results showed that these two methods yield comparable values. In addition, it was demonstrated that although primary induction produces changes in the cell that persist for periods of several months after the hormone is gone, these changes are not permanent. When Xenopus laevis withdrawn at least two years from estrogen are restimulated, the induction of vitellogenin mRNA follows the pattern observed in primary stimulation of male animals.
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