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dc.contributor.authorMallick, Shyamali
dc.identifier.citationSource: Dissertation Abstracts International, Volume: 57-10, Section: B, page: 6087.;Advisors: Susan Band Horwitz.
dc.description.abstractThe murine multidrug resistance gene mdr1b is highly induced in the endometrium during pregnancy. Induction occurs mainly as a result of progesterone action. Progesterone belongs to the family of steroid hormones whose receptors function as ligand-activated transcription factors that bind to their cognate response elements in the regulatory regions of many genes. 5{dollar}\sp\prime{dollar} flanking sequences between {dollar}-{dollar}540 and +97 of the mdr1b gene were fused to the reporter gene bacterial chloramphenicol acetyltransferase (p540CAT). In transient transfections, p540CAT was induced upon treatment with R5020, a progesterone analog, when cotransfected with the A form (PR{dollar}\sb{lcub}\rm A{rcub}{dollar}), but not the B form (PR{dollar}\sb{lcub}\rm B{rcub}{dollar}) of the receptor. p540CAT cotransfected with a DNA binding domain mutant of PR{dollar}\sb{lcub}\rm B{rcub}{dollar} was not induced. Coexpression of PR{dollar}\sb{lcub}\rm A{rcub}{dollar} and PR{dollar}\sb{lcub}\rm B{rcub}{dollar} inhibited the induction observed with PR{dollar}\sb{lcub}\rm A{rcub}{dollar} alone.;To determine whether mdr1b was an early or a late response gene for progesterone, a time course of activation was performed in HeLa cells transiently cotransfected with p540CAT and an expression vector for PR{dollar}\sb{lcub}\rm A{rcub}{dollar}. Activation was observed 12 hours after hormone addition and peaked at 18 hours suggestive of a late gene. Sequences between {dollar}-{dollar}234 and {dollar}-{dollar}206 which contain a partially conserved progesterone response element (mdrPRE) were modified to create a consensus PRE (cPRE) or mutated, to eliminate any similarity to a PRE (mPRE). In HeLa cells cotransfected with PR{dollar}\sb{lcub}\rm A{rcub}{dollar}, mPRE or wildtype were induced 4-fold while cPRE induced expression 8-fold. It suggests that mdrPRE, although partially conserved, does not function as a progesterone response element in this context. By gel shift analysis, the region containing mdrPRE does not bind any nuclear factors. Taken together, the mdr1b gene appears to be a late gene activated by indirect mechanisms.;Nested deletion analysis to identify sequences required for activation by progesterone was performed. Regions were designated A-E. Two regions D and E were further studied. Region D between {dollar}-{dollar}122 and {dollar}-{dollar}65 has previously been shown to bind C/EBP{dollar}\beta{dollar} and NF-Y and mutation of these binding sites decreased induction by progesterone. Region E contained sequences from {dollar}-{dollar}65 and including the first untranslated exon made a modest contribution to the overall progesterone response. In summary, mdr1b is a late gene and induction by progesterone occurs via a complex regulatory mechanism that depends on the factors C/EB{dollar}\beta{dollar} and NF-Y.
dc.publisherProQuest Dissertations & Theses
dc.subjectMolecular biology.
dc.titleStudies on the regulation of the murine multidrug resistance genemdr1b by progesterone

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