All ETDs from UAB

Advisory Committee Chair

Charles N Falany

Advisory Committee Members

John Parant

Timothy Kraft

Stephen Barnes

Lori L McMahon

Rosalinda Roberts

Document Type

Dissertation

Date of Award

2016

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Since its identification in 2000, sulfotransferase (SULT) 4A1 has presented an enigma to the field of cytosolic SULT biology. SULT4A1 is exclusively expressed in neural tissue, is highly conserved, and has been identified in every vertebrate studied to date. Despite this singular level of conservation, no substrate or function for SULT4A1 has been identified. Previous studies demonstrated that SULT4A1 does not bind the obligate sulfate donor, 3’-phosphoadenosine-5’-phosphosulfate (PAPS), yet SULT4A1 is classified as a SULT superfamily member based on sequence and structural similarities to the other SULTs. In this study, RNA-seq was used to search for alterations in gene ex-pression in 72 hours post fertilization zebrafish larvae following transient SULT4A1 knockdown (KD) utilizing splice blocking morpholino oligonucleotides (MOs). This study demonstrates that transient inhibition of SULT4A1 expression in developing zebrafish larvae results in the up-regulation of several genes involved in phototransduction. SULT4A1 KD was verified by immunoblot analysis and quantitative real-time PCR (qPCR). Gene regulation changes identified by deep RNA sequencing were validated by qPCR. Transcription activator-like effector nucleases (TALENs) were also used to gener-ate heritable mutations in the SULT4A1 gene of zebrafish. The mutation consists of an 8 nucleotide deletion within the second exon of the gene, resulting in a frameshift mutation and premature stop codon after 132 AA. During early adulthood, casual observations were made that mutant zebrafish were exhibiting excessively sedentary behavior during the day. These observations were inconsistent with published reports on activity in zebrafish which are largely diurnal organisms and are highly active during the day. Thus, a decrease in activity during the day represents an abnormal behavior and warranted fur-ther systematic analysis. EthoVision video tracking software was used to monitor activity levels in wild type and mutant fish over 48 hours of a normal light/dark cycle. SULT4A1 mutant fish were shown to exhibit increased inactivity bout length and frequency as well as a general decrease in daytime activity levels when compared to their WT counterparts.

Share

COinS