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dc.contributor.authorBautz, David Jamesen_US
dc.date.accessioned2014-03-14T20:39:09Z
dc.date.available2014-03-14T20:39:09Z
dc.date.issued2001-05-10en_US
dc.identifier.otheretd-06012001-143530en_US
dc.identifier.urihttp://hdl.handle.net/10919/33369
dc.description.abstractThe 5' end of eukaryotic and viral mRNAs contain a "cap" structure with the sequence m7G(5')pppN(5'). The methylation of the 7-position on the guanine cap is very important to proper mRNA processing and initiation of translation. The enzyme responsible for this methylation, RNA guanine-7-methyltransferase, has been cloned and studied from a number of different species, including human, X. laevis, yeast, and C. elegans. The sequences for mouse guanine-7-methyltransferase cDNA and protein have been deduced based upon identity of mouse ESTs to the cDNA of the human enzyme. The deduced mouse cDNA encodes an ORF of 465 amino acids and is 76.4% identical to the human enzyme, or 86.5% within the C-terminal domain. Active site characterization of mouse and human guanine-7-methyltransferase indicates a cysteine residue is important to proper enzyme activity. Enzyme activity was completely eliminated when N-ethylmaleimide (NEM) was added to the assay mixture. When the product of the reaction, S-adenosyl-L-homocysteine (SAH), was added at a concentration of 40uM the mouse enzyme retained 60% activity while enzyme isolated from Human Osteosarcoma (HOS) cells retained 100% of the original activity. SAH demonstrated no protective effects on the cloned human enzyme. Factors that affect binding of RNA to the active site were also investigated. UV-cross-linking of RNA to the active site of the mouse enzyme was inhibited 35% by NEM. Cap analog, GpppG, at a concentration of 1mM, inhibited cross-linking, but the similar nucleotide GMP, at a concentration of 1mM, did not inhibit cross-linking. These analyses have given a clearer understanding of this very important enzyme.en_US
dc.publisherVirginia Techen_US
dc.relation.haspartThesis6-02-01.pdfen_US
dc.rightsI hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to Virginia Tech or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report.en_US
dc.subjectGenomicsen_US
dc.subjectCap Structureen_US
dc.subjectNEMen_US
dc.subjectMethylationen_US
dc.titleGenomic Analysis of Human and Mouse Guanine-7-Methyltransferase with Active Site Characterizationen_US
dc.typeThesisen_US
dc.contributor.departmentBiochemistryen_US
dc.description.degreeMaster of Scienceen_US
thesis.degree.nameMaster of Scienceen_US
thesis.degree.levelmastersen_US
thesis.degree.grantorVirginia Polytechnic Institute and State Universityen_US
thesis.degree.disciplineBiochemistryen_US
dc.contributor.committeechairSitz, Thomas O.en_US
dc.contributor.committeememberGillaspy, Glenda E.en_US
dc.contributor.committeememberLuckhart, Shirleyen_US
dc.identifier.sourceurlhttp://scholar.lib.vt.edu/theses/available/etd-06012001-143530/en_US
dc.date.sdate2001-06-01en_US
dc.date.rdate2002-06-01
dc.date.adate2001-06-01en_US


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