Event Title

Non-Genotoxic Human APOBEC3A

Location

CSB Ballroom

Start Date

18-4-2016 10:00 AM

End Date

18-4-2016 11:30 AM

Student's Major

Biological Sciences

Student's College

Science, Engineering and Technology

Mentor's Name

Allison Land

Mentor's Department

Biological Sciences

Mentor's College

Science, Engineering and Technology

Description

APOBEC3A (A3A) is an enzyme that deaminates DNA cytosine to produce uracil. A3A plays a role in innate immunity to foreign DNA like HIV-1. HIV-1 counteracts APOBEC3 proteins with Vif, which targets the APOBEC3s for degradation. When A3A is naturally expressed in monocyte immune cells, it is retained in the cytoplasm. When A3A is transfected into cells, the localization is cell wide and it is able to mutate the genome. To circumvent this genotoxicity and mimic the natural scenario, a nuclear export sequence (NES) can be tacked onto A3A. A3A-NES has a cytoplasmic localization, and is non- genotoxic. The purpose of this research is to examine whether or not wild type and non- genotoxic human A3A are equally susceptible to Vif. I hypothesize that non-genotoxic versions of A3A are not equally degraded by Vif. To test my hypothesis, I will construct A3A constructs in the same plasmid background as APOBEC3G (A3G), an APOBEC3 protein known to be effectively neutralized by Vif. These plasmids will then be transfected into the human 293T cells with and without Vif. First, I used mutagenic primers to amplify A3G. Currently I am using the NotI and EcoRV restriction enzymes to cut my PCR amplicon and plasmid backbone. After I will ligate these pieces of DNA together. This product will be sequenced and maxi prepped to transfect into 293T cells. An immunoblot will be assessed, and I hope to see no significant cytotoxicity with A3A, and to find out that Vif helps decrease A3A levels.

This document is currently not available here.

Share

COinS
 
Apr 18th, 10:00 AM Apr 18th, 11:30 AM

Non-Genotoxic Human APOBEC3A

CSB Ballroom

APOBEC3A (A3A) is an enzyme that deaminates DNA cytosine to produce uracil. A3A plays a role in innate immunity to foreign DNA like HIV-1. HIV-1 counteracts APOBEC3 proteins with Vif, which targets the APOBEC3s for degradation. When A3A is naturally expressed in monocyte immune cells, it is retained in the cytoplasm. When A3A is transfected into cells, the localization is cell wide and it is able to mutate the genome. To circumvent this genotoxicity and mimic the natural scenario, a nuclear export sequence (NES) can be tacked onto A3A. A3A-NES has a cytoplasmic localization, and is non- genotoxic. The purpose of this research is to examine whether or not wild type and non- genotoxic human A3A are equally susceptible to Vif. I hypothesize that non-genotoxic versions of A3A are not equally degraded by Vif. To test my hypothesis, I will construct A3A constructs in the same plasmid background as APOBEC3G (A3G), an APOBEC3 protein known to be effectively neutralized by Vif. These plasmids will then be transfected into the human 293T cells with and without Vif. First, I used mutagenic primers to amplify A3G. Currently I am using the NotI and EcoRV restriction enzymes to cut my PCR amplicon and plasmid backbone. After I will ligate these pieces of DNA together. This product will be sequenced and maxi prepped to transfect into 293T cells. An immunoblot will be assessed, and I hope to see no significant cytotoxicity with A3A, and to find out that Vif helps decrease A3A levels.

Recommended Citation

Hadler, Jessica. "Non-Genotoxic Human APOBEC3A." Undergraduate Research Symposium, Mankato, MN, April 18, 2016.
http://cornerstone.lib.mnsu.edu/urs/2016/poster-session-A/19