Faculty Advisor or Committee Member

Kristin Wobbe, Advisor

Faculty Advisor or Committee Member

Jose Arguello, Committee Member

Identifier

etd-082206-113216

Abstract

"Host resolution of viral infection is dependent upon components of the innate and acquired immune system. The mammalian protein Apobec3 plays an important role as part of the immune system’s innate defenses through its modification of reverse transcribed viral DNA. Recently, Apobec3 was found to directly inhibit HIV-1 and HBV replication through deaminating newly transcribed deoxycytidine residues to deoxyuridine. The ability of mouse and simian Apobec3 variants to inhibit human retroviruses and vice versa highlights the utility of analyzing cross-species homologues. To better understand this editing enzyme, differentially pathogen-susceptible inbred mice were used as an experimental model. The purpose of this project is to examine the effects of murine Apobec3 (muA3) alternative splicing on its DNA-editing characteristics. Three distinct Apobec3 isoforms were isolated from pathogen-susceptible BALB/cByJ (“C”) inbred mice, and two Apobec3 isoforms came from pathogen-resistant C57BL/6ByJ (“Y”) mice. The five muA3 isoforms were cloned, sequenced, and expressed from a constitutive promoter in a haploid Saccharomyces cerevisia strain. MuA3 DNA-editing activity was measured via the CAN1 forward mutation assay. The five isoforms studied in this project were discovered to be strain-specific. One isoform from each mouse strain mutated the yeast CAN1 locus significantly. Additionally, both muA3 isoform mRNAs derived from the pathogen-resistant Y mice were found to persist at a higher level (2.7 -12.4 fold) than any of the C mouse isoforms. This suggests that the absence of exon 5 or some other signal in the Y mice may influence transcript stability. Evidence also suggests that the murine Apobec3 start codon is actually 33bp upstream of its reference start, with implications for previous research performed using muA3. Sequencing analysis of genomic DNA revealed the presence of a 4bp insertion in a region of BALB/cByJ muA3 which may have disrupted an intronic splicing enhancer signal. Furthermore, a novel BALB/cByJ Apobec3 isoform was characterized. This is the first report of strain-specific processing with regard to muA3."

Publisher

Worcester Polytechnic Institute

Degree Name

MS

Department

Chemistry & Biochemistry

Project Type

Thesis

Date Accepted

2006-08-22

Accessibility

Unrestricted

Subjects

gene function and regulation, splicing, apobec3, genetics, Antiretroviral agents, Genetic regulation

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