Publication Date

2017-08-10

Availability

Embargoed

Embargo Period

2019-08-10

Degree Type

Thesis

Degree Name

Master of Science (MS)

Department

Marine Biology and Fisheries (Marine)

Date of Defense

2017-07-21

First Committee Member

Michael Schmale

Second Committee Member

Nevis L. Fregien

Third Committee Member

Marjorie Oleksiak

Abstract

Damselfish Neurofibromatosis (DNF) is a naturally occurring cancer that affects Bicolor Damselfish (Stegastes partitus) located on South Florida reefs. Damselfish Neurofibromatosis is characterized by tumor development within the pigmented cells of the skin (chromatophoromas) as well as Schwann cells (neurofibromas and malignant peripheral nerve sheath tumors). Damselfish Neurofibromatosis has been shown to be caused by a virus-like agent with a 2.4 kb DNA genome named the Damselfish Virus-like Agent (DVLA). Studies of DVLA have shown that this DNA is located and replicates within the mitochondria of infected cells. Five RNAs are transcribed from the DVLA genome but the mechanism of how the genome is transcribed remains unknown. The purpose of this study was to further investigate how the DNA genome of DVLA is transcribed within the cells and how this ultimately affects the tumorigenesis of the virus-like agent. Primer sets were designed which recognize all five transcripts (DVLA stem) or only the longest transcript (DVLA loop) of DVLA. Inhibition of RNA polymerase II with triptolide caused significantly more inhibition of nuclear transcripts than of DVLA stem and the mitochondrial transcripts. However, DVLA loop showed no significant difference in inhibition from either the nuclear or mitochondrial transcripts. Treatment with ethidium bromide to block mitochondrial RNA transcription caused significantly greater inhibition of the DVLA and mitochondrial transcripts compared to the nuclear transcripts. These results are consistent with the transcription of DVLA RNAs by mitochondrial rather than nuclear polymerases. DVLA loop had a strong positive correlation with cytochrome-b and D-loop in total RNA levels. DVLA loop had a short half-life, high synthesis rate, and low copy number count similar to D-loop. These similarities suggest that DVLA loop may serve a similar role as D-loop in RNA transcription and the initiation of DNA replication. Transcripts identified with DVLA stem primers had the longest half-life of the transcripts tested despite being non-polyadenylated, suggesting the presence of an alternative method for stabilizing these RNAs.

Keywords

Bicolor Damselfish; RNA; Transcription; Virus-like agent; Cancer; 4sU labeling

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