Document Type

Open Access Thesis

Embargo Period

8-29-2016

Degree Program

Microbiology

Degree Type

Master of Science (M.S.)

Year Degree Awarded

2016

Month Degree Awarded

May

Advisor Name

Michele

Advisor Middle Initial

M

Advisor Last Name

Klingbeil

Co-advisor Name

Steve

Co-advisor Last Name

Sandler

Third Advisor Name

Yasu

Third Advisor Last Name

Morita

Abstract

Trypanosoma brucei is the causative agent of Human African Trypanosomiasis (HAT), also known as African sleeping sickness. T. brucei is unique in several ways that distinguish this organism from other eukaryotes. One of the unique features of T. brucei is the organism’s mitochondrial DNA, which is organized in a complex structure called kinetoplast DNA (kDNA). Since kDNA is unique to the kinetoplastids, kDNA may serve as a good drug target against T. brucei. Previews studies have shown that kDNA has 4 different family A mitochondrial DNA polymerases. Three of these mitochondrial DNA polymerases (POLIB, POLIC, and POLID) are essential components of kDNA synthesis and replication. POLID and POLIC dynamically localize throughout the cell cycle. POLID is found dispersed in the matrix before the kDNA has undergone replication and is re-localized at the antipodal sites when the kDNA is dividing. POLIC is found in the kinetoflagellar zone (KFZ) at low concentrations when the kDNA is not replicating and relocalizes to the antipodal sites when dividing. Based on the dynamic localization of these two DNA polymerases, we hypothesize that POLIB undergoes dynamic localization at some point during the cell cycle stage. Here, a POLIB/PTP single expressor cell line was analyzed by immunofluorescence microscopy in an unsynchronized population. We characterized the localization pattern of POLIB-PTP at different cell cycle stages and found different localization patterns throughout cell cycle. Cells at 1N1K (the majority of cell in an unsynchronized population) have single foci, but at 1N1Kdiv two different patterns are mainly observed, diffuse and segregated. When the kDNAs are separated POLIB-PTP is again seen as a distinct foci in each kDNA. By doing TdT labeling and a quantitative analysis, we found that at early stages of minicircles replication POLIB-PTP start relocalizing to the kDNA disk with a diffuse pattern being the main. By the time the minicircles are being reattached in the disk (late TdT), POLIB is seen in the disk as a bilobe shape.

First Advisor

Michele Klingbeil

Second Advisor

Steve Sandler

Third Advisor

Yasu Morita

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