Document Type

Thesis

Date of Award

7-1997

School/College

College of Science, Engineering, and Technology (COSET)

Degree Name

MS in Biology

First Advisor

Yvonne H. Hogan

Abstract

Orphan receptors form an important subfamily of the nuclear receptor superfamily. They are identified on the basis of homology in the DNA binding domain to previously cloned members. These novel members of the superfamily are named orphan receptors because of their unknown biological functions and putative ligands. A novel member of the nuclear receptor superfamily termed Germ Cell Nuclear Factor (GCNF) has been recently cloned. It is expressed predominantly in the male and female germ cells of the adult mouse. Analysis was undertaken to shed light on GCNF's transactivation properties and to identify target responsive genes. It has been show that GCNF binds to response elements which are configured as direct repeats of the consensus half site AGGTCA with a 0 bp spacing and that it binds to this element as a homodimer. A computer search of Genbank identified a number of potential responsive genes containing DRO sequences, one of which was the Lactoferrin gene. Transcription of the Lactoferrin gene 1 2 is stimulated by estrogen in the mouse uterus. Analysis of the promoter revealed an estrogen responsive element (ERE) overlapping with a chicken ovalbumin up-stream promoter (COUP) element located at position -349 to -329 from the transcription initiation site. The ERE element differed from the consensus Ekf sequences by one nucleotide at the second position of the 3' half of the element (G to A); the COUP element differed by one nucleotide from the consensus COUP element. The ERE} COUP element confers estrogen action to both homologous and heterologous promoters. Competition experiments with ERE and COUP oligonucleotides have been performed to confirm interaction between Lactoferrin ERE / COUP element with the COUP transcription factor and the estrogen receptor, respectively; and this sequence is a complex steroid response element and was renamed mERM for mouse lactoferrin estrogen response module. COUP-TF represses the promoter through this element. In knockout mice of the Lactoferrin gene, pre-implantation embryonic lethality is observed. The Lactoferrin gene is not expressed maternally in the oocyte; however, the Lactoferrin gene is turned on at the 2 cell and 4 cell stage of embryogenesis. Lactoferrin expression is maintained to the 32 cell stage. In the Lactoferrin ERM, four half sites are juxtaposed to generate a number of repeats; an IR3, which is the ERE, DRO to which GCNF may bind, and DRI to which COUP-TF binds. All of these half sites are 1 nucleotide away from a perfect AGGTCA core sequence except the central half site which is perfect. In this study, experiments were performed to determine whether GCNF binds to the promoter region in the Lactoferrin (targeting the ERM). Other experiments performed were dose-dependent binding of GCNF to the WT mERM, 3 competition with the WT mERM and PRE to establish specificity of binding, and binding of GCNF to mDR1, mDRO, mERE and mERM mutant sequences

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