Abstract
We report on the isolation and characterization of cDNA and genomic sequences encoding a chicken cardiac phospholamban. Three mRNAs, 0.6, 1.1, and 3.3 kilobase pairs in size, were detected in cardiac and slow-tonic muscle RNAs on Northern blots. We determined that these mRNAs differ in the length of their 3'-untranslated regions, perhaps because they utilize alternative polyadenylation signals. We studied the developmental and tissue-specific expression of phospholamban mRNA using nuclease mapping analysis. There appeared to be considerable homology between coding and 3'-untranslated regions of phospholamban mRNAs from cardiac and slow-tonic muscles during development, strongly suggesting that these mRNAs are encoded by the same gene. The phospholamban gene, which is present as a single copy in the chicken genome, is about 10 kilobase pairs long, with approximately 6.5 kilobase pairs representing the intron sequences. Primer extension and nuclease mapping analyses of the 5' end of the phospholamban mRNA showed that the three transcripts are initiated from the same initiation site. Analysis of the putative promoter region revealed "TATA box" and "CAAT box" sequences, putative muscle-specific elements, and a cyclic AMP-responsive element.
Highlights
From the Departmentof $§Medicine, §Anatomy, and §Pharmacological and Physiological Sciences, The University of Chicago, Chicago, Illinois 60637
We report on the isolation and characterization of identical subunits, each composed of two major domains: an cDNA and genomic sequences encoding a chicken car- a-helical domain, which has phosphorylated sites, and a hydiac phospholamban
It hasbeen proposed that phospholamban existsas a pentamer of identical subunits thartegulates Ca”-ATPase activity, anchoring its hydrophobic region into the sarcoplasmic reticulum membrane.Inthisstudy, we have isolated the cDNA and the gene encoding a chicken phospholamban
Summary
From the Departmentof $§Medicine, §Anatomy, and §Pharmacological and Physiological Sciences, The University of Chicago, Chicago, Illinois 60637. A direct interackilobase pairs in size, were detected in cardiac and slow-tonic muscle RNAs on Northern blots We determined that these mRNAs differ in the length of their 3”untranslated regions, perhaps because they utilize alternative polyadenylation signals. To evaluate the structure-function relationshipand the processes that control the tissue specificity and transcriptional regulation of the phospholamban gene during development, we first isolated and characterized the cDNA clones that encode a chicken cardiac phospholamban. ATPase of cardiac muscle sarcoplasmic reticulum [1,2] This When we used region-specific cDNAs as probes for Northern enzyme is responsible for the rapid removal of Ca2+ ionsfrom blot analysis, various sized transcripts appeared to represent the cytosol, which is necessary for the efficient relaxation of mRNAs having different lengths of 3”untranslated regions. Maryland Ave., Chicago, chased from Amersham Corp Cloning and Isolation of Genomic DNA Clones-We used a chicken separation were carried out asdescribed above
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