{"id":{"repo_id":"vt","oai_identifier":"oai:vtechworks.lib.vt.edu:10919/33369"},"canonical_url":"https://search.dev.ndltd.org/etd/vt/oai:vtechworks.lib.vt.edu:10919/33369","repository":{"repo_id":"vt","name":"Virginia Tech","base_url":"https://vtechworks.lib.vt.edu/oai/request"},"display":{"title":"Genomic Analysis of Human and Mouse Guanine-7-Methyltransferase with Active Site Characterization","abstract":"The 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.","abstract_html":"The 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.","abstract_has_math":false,"creators":["Bautz, David James"],"institution":"Virginia Tech","degree_name":"Master of Science","degree_level":"masters","degree_discipline":"Biochemistry","degree_department":"Biochemistry","school":null,"contributors":[],"advisors":[],"committee_chairs":["Sitz, Thomas O."],"committee_members":["Gillaspy, Glenda E.","Luckhart, Shirley"],"year":2001,"date_issued":"2001-05-10","date_published":"2001-05-10","updated_at":"2026-07-22T22:19:47Z","subjects":["Genomics","Cap Structure","NEM","Methylation"],"languages":[],"rights":["In Copyright"],"rights_urls":["http://rightsstatements.org/vocab/InC/1.0/"],"identifier_entries":[{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["etd-06012001-143530"],"render_values":[{"text":"etd-06012001-143530","href":null,"code":true}]}]},"links":{"outbound_url":"http://hdl.handle.net/10919/33369","outbound_label":"Handle","outbound_source":"dc:identifier.uri"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor.committeechair","label":"Committee Chair","values":["Sitz, Thomas O."]},{"key":"dc:contributor.committeemember","label":"Committee Member","values":["Gillaspy, Glenda E.","Luckhart, Shirley"]},{"key":"dc:contributor.department","label":"Department","values":["Biochemistry"]},{"key":"dc:creator","label":"Author","values":["Bautz, David James"]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.accessioned","label":"Dc Date Accessioned","values":["2014-03-14T20:39:09Z"]},{"key":"dc:date.available","label":"Dc Date Available","values":["2014-03-14T20:39:09Z","2002-06-01"]},{"key":"dc:date.issued","label":"Date","values":["2001-05-10"]},{"key":"dc:publisher","label":"Institution","values":["Virginia Tech"]},{"key":"dc:type","label":"Dc Type","values":["Thesis"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Biochemistry"]},{"key":"thesis:degree_level","label":"Degree Level","values":["masters"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Master of Science"]},{"key":"thesis:institution_name","label":"Thesis Institution Name","values":["Virginia Polytechnic Institute and State University"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Genomics","Cap Structure","NEM","Methylation"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:rights","label":"Dc Rights","values":["In Copyright"]},{"key":"dc:rights.uri","label":"Rights URI","values":["http://rightsstatements.org/vocab/InC/1.0/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier.other","label":"Dc Identifier Other","values":["etd-06012001-143530"]},{"key":"dc:identifier.uri","label":"Identifier URI","values":["http://hdl.handle.net/10919/33369"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["The 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."]},{"key":"dc:description.degree","label":"Dc Description Degree","values":["Master of Science"]},{"key":"dc:title","label":"Title","values":["Genomic Analysis of Human and Mouse Guanine-7-Methyltransferase with Active Site Characterization"]}]}],"canonical_facts":{"dc:contributor.committeechair":["Sitz, Thomas O."],"dc:contributor.committeemember":["Gillaspy, Glenda E.","Luckhart, Shirley"],"dc:contributor.department":["Biochemistry"],"dc:creator":["Bautz, David James"],"dc:date.accessioned":["2014-03-14T20:39:09Z"],"dc:date.available":["2014-03-14T20:39:09Z","2002-06-01"],"dc:date.issued":["2001-05-10"],"dc:description.abstract":["The 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."],"dc:description.degree":["Master of Science"],"dc:identifier.other":["etd-06012001-143530"],"dc:identifier.uri":["http://hdl.handle.net/10919/33369"],"dc:publisher":["Virginia Tech"],"dc:rights":["In Copyright"],"dc:rights.uri":["http://rightsstatements.org/vocab/InC/1.0/"],"dc:subject":["Genomics","Cap Structure","NEM","Methylation"],"dc:title":["Genomic Analysis of Human and Mouse Guanine-7-Methyltransferase with Active Site Characterization"],"dc:type":["Thesis"],"thesis:degree_discipline":["Biochemistry"],"thesis:degree_level":["masters"],"thesis:degree_name":["Master of Science"],"thesis:institution_name":["Virginia Polytechnic Institute and State University"]},"updated_at":"2026-07-22T22:19:47Z"}