Molecular cloning, characterization, and regulation of diacylglycerol kinases

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Title Molecular cloning, characterization, and regulation of diacylglycerol kinases
Publication Type dissertation
School or College School of Medicine
Department Biochemistry
Author Ding, Li
Date 1998-08
Description Diacylglycerol (DAG) is a key intermediate in the synthesis of complex lipids and serves as an important second messenger. DAG exerts its signaling function by activation protein kinase C (PKC). While ending the signals(s) from DAG, the diacylglycerol kinase (DGK) reaction generates phosphatidic acid (PA) that is potentially mitogenic. Besides its effects on DAG signaling, the DAK reaction is the first step in the recycling of phosphatidylinositol species following their hydrolysis. Two DGKzeta, cDNAs, differed from the 3.5 kb clone identified in endothelial cells, were isolated from a human skeletal muscle cDNA library. One transcript, 3.4 kb in length, was shown to be nonfunctional. The other, 4.1 kb in length, has a unique 5' sequence of 853 bp. The DGKzeta gene contains 32 exons and approximately 50 kb of genomic sequence. The protein encoded by the 4.1 kb transcript contains a unique N-terminal domain. The coding sequence was shown to be derived from alternative splicing of the DGKzeta gene. In cells transfected with 4.1 kb clone, we detected a 130 kDa protein with an antibody to DGKzeta and demonstrated that it was localized predominantly in the nucleus. The expression of DGKzeta protein increases gradually in embryos from E10.5 to E14.5. The brains of both newborn and adult mice have abundant DGKzeta protein. The location of DGK kinases mRNA id developing embryos was determined by RNA whole-mount in situ hybridization. It is highly expressed in the sensory nerve system, for example, the dorsal root ganglia and vibrissa follicles. By using the human DGKzeta sequence as the query sequence, we uncover an EST clone from a human retina library. DGK? was subsequently isolated using this EST clone as a probe. DGK? contains two cysteine-rich repeats, a myristoylated alanine-rich c kinase substrate (MARCKS) site, a conserved catalytic domain and four ankyrin repeats at it C-terminus, displaying great similarity to both human DGK kinases and Drosophila DGK2, rdgA. A DGK? is expressed in brain and retina among tissues examined. DGK? encodes a 130 kDa protein and localizes in both cytoplasmic and nuclear fractions. The RR10 form of autosomal dominant retinitis pigmentosa (adRP) was mapped to a 5-centimorgan (cM) region on human chromosome 7q31. Human DGK? was mapped to chromosome 7q32.3 close to the RP10 locus. A potential disease-causing mutation was searched for in an American RP10 family (UTAD045) using single strand conformation polymorphism (SSCP) and direct genomic sequencing. Sequencing of the polymerase chain reaction (PCR) products of 31 DGK? exons from the patients reveled no disease-causing mutations but polymorphisms. By using a polymorphism in the DGK? gene as a marker, a recombinant was found in this family. These results excluded DGK? as the causative gene for this form of RP10.
Type Text
Publisher University of Utah
Subject Protein kinases
Subject MESH Protein Kinases
Dissertation Institution University of Utah
Dissertation Name PhD
Language eng
Relation is Version of Digital reproduction of "The molecular cloning, characterization, and regulation of diacylglycerol kinases". Spencer S. Eccles Health Sciences Library.
Rights Management © Li Ding.
Format application/pdf
Format Medium application/pdf
Format Extent 2,208,547 bytes
Identifier undthes,4265
Source Original: University of Utah Spencer S. Eccles Health Sciences Library (no longer available).
Master File Extent 2,208,563 bytes
ARK ark:/87278/s6gh9kqw
Setname ir_etd
ID 190757
Reference URL https://collections.lib.utah.edu/ark:/87278/s6gh9kqw
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