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dc.contributor.authorHu, Erding
dc.identifier.citationSource: Dissertation Abstracts International, Volume: 52-02, Section: B, page: 6550.;Advisors: Charles S. Rubin.
dc.description.abstractCasein kinase II (CKII) is a ubiquitous, eukaryotic Ser/Thr protein kinase that has been implicated in many biological processes including growth factor signal transduction, transcription activation, and oncogenesis. Our goal is to use nematode C. elegans as a model to study the structure, function, and regulation of the enzyme.;C. elegans CKII was purified to homogeneity and shown to contain {dollar}\alpha{dollar}(42 kDa) and {dollar}\beta{dollar}(29 kDa) subunits. Cytosols from C. elegans embryos and gravid adults, are enriched in CKII activity (3-10 fold), as compared with other stages of development, suggesting a major role for CKII in C. elegans early development. cDNAs that encode CKII{dollar}\alpha{dollar} and {dollar}\beta{dollar} subunits were cloned and sequenced. Northern analysis revealed that the CKII{dollar}\alpha{dollar} and {dollar}\beta{dollar} mRNA levels vary in accord with the changes in enzyme activity during C. elegans development. Sequence analysis of the CKII{dollar}\alpha{dollar} and {dollar}\beta{dollar} gene clones elucidated the complete genomic organization for both subunits.;CKII{dollar}\alpha{dollar} cDNA was inserted into a bacterial expression system to generate soluble, catalytically active enzyme. Purified rCKII{dollar}\alpha{dollar} exhibits many properties that are characteristic of the CKII holoenzyme, but the k{dollar}\sb{lcub}\rm cat{rcub}{dollar} for rCKII{dollar}\alpha{dollar} was only 9% of that measured for holoenzyme, suggesting a possible role for {dollar}\beta{dollar} in CKII activation. The rCKII{dollar}\alpha{dollar} is heparin sensitive, indicating that this is an intrinsic property of the {dollar}\alpha{dollar} subunit. To further define the heparin-sensitive, inhibitory domain, a highly-conserved polybasic region(residues 73-82) in the {dollar}\alpha{dollar} subunit was modified by site-directed mutagenesis. Several rCKII{dollar}\alpha{dollar} mutants that exhibited decreased sensitivity to heparin were isolated. The most striking change was obtained when two Lys residues in the polycationic stretch were substituted with Glu. This caused a 70-fold decline in the affinity of the catalytic subunit for heparin.;Since the 1.7kb mRNA for CKII{dollar}\alpha{dollar} is most abundant in embryos, efforts were made to identify developmentally-regulated proteins that interact with the 5'-flanking region of the CKII{dollar}\alpha{dollar} gene. Using gel shift and DNase I protection assays, I have identified a DNA binding factor from extracts of C. elegans. The factor binds to a novel target sequence in the CKII{dollar}\alpha{dollar} gene promotor, and the binding activity is highly enriched ({dollar}>{dollar}10x) in C. elegans embryos. A Southwestern blot experiment indicated that the DNA binding activity is due to a 75 kDa protein.
dc.publisherProQuest Dissertations & Theses
dc.subjectMolecular biology.
dc.titleCasein kinase II from Caenorhabditis elegans: Structure, function and regulation

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