Date of Award

1997

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Molecular and Cellular Biology and Pathobiology

College

College of Graduate Studies

Abstract

The sodium-calcium exchanger (NCX1) plays a major role in calcium efflux, and therefore, in calcium homeostasis in the heart. The exchanger mRNA and protein is rapidly upregulated within one hour of pressure overload. In this work, the exchanger has been shown to be upregulated by persistent pressure overload for at least one to two weeks. The 5' end of the NCX1 gene has been characterized in order to examine transcriptional regulation of the cardiac promoter. Four feline NCX1 isoforms of the 5' untranslated region have been cloned: one from brain (Br1), one from kidney (K1), and two from heart (H1 and H2). Each isoform is expressed in a tissue specific manner. Seven unique clones of the alternatively spliced portion of the NCX1 intracytoplasmic loop have been identified in libraries from feline heart, brain, liver and kidney. These also exhibit tissue specific expression. Three are unique to brain. Three clones are expressed in kidney; one of these three is exclusively expressed in fetal kidney. Two isoforms are expressed in heart: one is unique to heart, and the other is predominately expressed in kidney. Both cardiac exchanger loop isoforms are upregulated for at least one to two weeks under continuous pressure overload. The 5'-end of the feline NCX1 gene has been cloned. The four tissue specific 5'untranslated regions are encoded by three alternatively spliced exons that have been mapped to nonoverlapping genomic clones. Exons K1 and Br1 lie on the same 13 Kb genomic clone separated by approximately 1 Kb. H1 has been mapped to a separate upstream 15 Kb genomic clone. Exon 2, the first common exon which encodes the translational start site, is found on a separate downnstream 17 Kb genomic clone. The K1 and Br1 isoforms each have unique promoters. The two heart isoforms, H1 and H2, share a single cardiac promoter. Primer extension has been used to map three transcriptional start sites for the cardiac promoter at 131, 139, and 143 bp 5' of the AUG. These sites have been confirmed by S1 nuclease protection assays. A DNA fragment containing 2000 bp of cardiac 5' flanking region, exon HI, and 67 bp of intron drives expression in cardiocytes, but not mouse L cells. Through deletion analysis, a fragment of the 5' flanking region from -184 to +200 has been determined to be the minimal promoter sufficient to drive expression and alpha-adrenergic induction in cardiocytes. At least one enhancer has been detected between -2000 and 1054, and another between +22 and + 112. One or more negative elements exist between -1054 and -184. Further study will lead to identification of specific positive and negative elements involved in transcriptional control of the NCX1 gene.

Rights

All rights reserved. Copyright is held by the author.

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