The primary focus of this study was on the characterization of the 122 nucleotide 5' Untranslated Region (UTR) of the Alcohol Oxidase I (AOXI) gene in Pichia pastoris. The 5' UTR influences the expression of many heterologous proteins in P. pastoris. However, no systematic analysis has ever been performed on this region to date. Several truncated versions of the 5' UTR were constructed using the QuikChange II XL Site Directed Mutagenesis Kit from Stratagene, PCR, and primers designed for a distinct region. Deletions of 21, 25, 30, 43, 61, 78, and 95 nucleotides were done to the 5' UTR. Elongated versions of the 5' UTRs were constructed where fragments of 10, 20, 30, 33, 36, 40, 45, and 50 nucleotides were inserted into the vector, subsequently increasing the length of the 5' UTR. All constructs were assessed using the β-galactosidase activity assay to determine if various constructs led to an increase or decrease in the rate of translation. Deletions had a variable effect on β-galactosidase expression, whereas additions decreased expression but not in a linear fashion. Final confirmation was performed using Northern analysis to ensure that the effects were due to translation rates and not nRNA transcription or degradation.
","abstract_html":"<p>The primary focus of this study was on the characterization of the 122 nucleotide 5' Untranslated Region (UTR) of the Alcohol Oxidase I (AOXI) gene in Pichia pastoris. The 5' UTR influences the expression of many heterologous proteins in P. pastoris. However, no systematic analysis has ever been performed on this region to date. Several truncated versions of the 5' UTR were constructed using the QuikChange II XL Site Directed Mutagenesis Kit from Stratagene, PCR, and primers designed for a distinct region. Deletions of 21, 25, 30, 43, 61, 78, and 95 nucleotides were done to the 5' UTR. Elongated versions of the 5' UTRs were constructed where fragments of 10, 20, 30, 33, 36, 40, 45, and 50 nucleotides were inserted into the vector, subsequently increasing the length of the 5' UTR. All constructs were assessed using the β-galactosidase activity assay to determine if various constructs led to an increase or decrease in the rate of translation. Deletions had a variable effect on β-galactosidase expression, whereas additions decreased expression but not in a linear fashion. Final confirmation was performed using Northern analysis to ensure that the effects were due to translation rates and not nRNA transcription or degradation.</p>","abstract_has_math":false,"creators":["Staley, Christopher A."],"institution":null,"degree_name":"Master of Science (M.S.)","degree_level":"Thesis - Pacific Access Restricted","degree_discipline":"Biological Sciences","degree_department":null,"school":null,"contributors":["Geoff Lin-Cereghino"],"advisors":[],"committee_chairs":[],"committee_members":[],"year":2007,"date_issued":"2007-01-01T08:00:00Z","date_published":"2007-01-01T08:00:00Z","updated_at":"2026-07-24T05:36:35Z","subjects":["Pichia pastoris","Genetic transcription","Yeast fungi Genetic engineering","Recombinant proteins","Life Sciences"],"languages":[],"rights":[],"rights_urls":["http://rightsstatements.org/vocab/InC/1.0/"],"identifier_entries":[]},"links":{"outbound_url":"https://scholarlycommons.pacific.edu/uop_etds/646","outbound_label":"Repository record","outbound_source":"dc:identifier"},"metadata_groups":[{"id":"people","label":"People","entries":[{"key":"dc:contributor","label":"Contributor","values":["Geoff Lin-Cereghino"]},{"key":"dc:creator","label":"Author","values":["Staley, Christopher A."]}]},{"id":"academic_context","label":"Academic Context","entries":[{"key":"dc:date.available","label":"Dc Date Available","values":["2007-01-01T08:00:00Z"]},{"key":"thesis:degree_discipline","label":"Discipline","values":["Biological Sciences"]},{"key":"thesis:degree_level","label":"Degree Level","values":["Thesis - Pacific Access Restricted"]},{"key":"thesis:degree_name","label":"Degree Name","values":["Master of Science (M.S.)"]}]},{"id":"subjects_keywords","label":"Subjects and Keywords","entries":[{"key":"dc:subject","label":"Dc Subject","values":["Pichia pastoris","Genetic transcription","Yeast fungi Genetic engineering","Recombinant proteins","Life Sciences"]}]},{"id":"language_rights","label":"Language and Rights","entries":[{"key":"dc:rights","label":"Dc Rights","values":["http://rightsstatements.org/vocab/InC/1.0/"]}]},{"id":"identifiers","label":"Identifiers","entries":[{"key":"dc:identifier","label":"Identifier","values":["https://scholarlycommons.pacific.edu/uop_etds/646"]}]},{"id":"additional","label":"Additional Metadata","entries":[{"key":"dc:description.abstract","label":"Abstract","values":["The primary focus of this study was on the characterization of the 122 nucleotide 5' Untranslated Region (UTR) of the Alcohol Oxidase I (AOXI) gene in Pichia pastoris. The 5' UTR influences the expression of many heterologous proteins in P. pastoris. However, no systematic analysis has ever been performed on this region to date. Several truncated versions of the 5' UTR were constructed using the QuikChange II XL Site Directed Mutagenesis Kit from Stratagene, PCR, and primers designed for a distinct region. Deletions of 21, 25, 30, 43, 61, 78, and 95 nucleotides were done to the 5' UTR. Elongated versions of the 5' UTRs were constructed where fragments of 10, 20, 30, 33, 36, 40, 45, and 50 nucleotides were inserted into the vector, subsequently increasing the length of the 5' UTR. All constructs were assessed using the β-galactosidase activity assay to determine if various constructs led to an increase or decrease in the rate of translation. Deletions had a variable effect on β-galactosidase expression, whereas additions decreased expression but not in a linear fashion. Final confirmation was performed using Northern analysis to ensure that the effects were due to translation rates and not nRNA transcription or degradation.
"]},{"key":"dc:source","label":"Dc Source","values":["82"]},{"key":"dc:title","label":"Title","values":["Characterization of the 5̕ untranslated region ( 5̕ UTR) of the alcohol oxidase I (AOX I) gene in Pichia pastoris."]}]}],"canonical_facts":{"dc:contributor":["Geoff Lin-Cereghino"],"dc:creator":["Staley, Christopher A."],"dc:date.available":["2007-01-01T08:00:00Z"],"dc:description.abstract":["The primary focus of this study was on the characterization of the 122 nucleotide 5' Untranslated Region (UTR) of the Alcohol Oxidase I (AOXI) gene in Pichia pastoris. The 5' UTR influences the expression of many heterologous proteins in P. pastoris. However, no systematic analysis has ever been performed on this region to date. Several truncated versions of the 5' UTR were constructed using the QuikChange II XL Site Directed Mutagenesis Kit from Stratagene, PCR, and primers designed for a distinct region. Deletions of 21, 25, 30, 43, 61, 78, and 95 nucleotides were done to the 5' UTR. Elongated versions of the 5' UTRs were constructed where fragments of 10, 20, 30, 33, 36, 40, 45, and 50 nucleotides were inserted into the vector, subsequently increasing the length of the 5' UTR. All constructs were assessed using the β-galactosidase activity assay to determine if various constructs led to an increase or decrease in the rate of translation. Deletions had a variable effect on β-galactosidase expression, whereas additions decreased expression but not in a linear fashion. Final confirmation was performed using Northern analysis to ensure that the effects were due to translation rates and not nRNA transcription or degradation.
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