Campus Access Only

All rights reserved. This publication is intended for use solely by faculty, students, and staff of University of the Pacific. No part of this publication may be reproduced, distributed, or transmitted in any form or by any means, now known or later developed, including but not limited to photocopying, recording, or other electronic or mechanical methods, without the prior written permission of the author or the publisher.

Date of Award

2007

Document Type

Thesis - Pacific Access Restricted

Degree Name

Master of Science (M.S.)

Department

Biological Sciences

First Advisor

Geoff Lin-Cereghino

First Committee Member

Craig Vierra

Second Committee Member

Lisa Wrischnik

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.

Pages

82

To access this thesis/dissertation you must have a valid pacific.edu email address and log-in to Scholarly Commons.

Find in PacificSearch

Share

COinS

If you are the author and would like to grant permission to make your work openly accessible, please email

 

Rights Statement

Rights Statement

In Copyright. URI: http://rightsstatements.org/vocab/InC/1.0/
This Item is protected by copyright and/or related rights. You are free to use this Item in any way that is permitted by the copyright and related rights legislation that applies to your use. For other uses you need to obtain permission from the rights-holder(s).