A Novel Third Isoform of Zebrafish Cytochrome

Oxidase IV Brandon Smith

Dr. Nancy Bachman, Faculty Advisor

Mitochondria

• Mitochondria are the energy production sites in the cell.

• Within the mitochondria, specific complexes of proteins carry out chemical reactions.

• These chemical reactions eventually lead to the production of ATP.

Role of Cytochrome Oxidase• Cytochrome Oxidase (COX) is the last enzyme in

the mitochondrial respiratory chain.• COX IV is thought to have a regulatory function

in energy production. This subunit has a number 2 different isoforms.

• Mammals have two isoforms for COX IV – a widely expressed form and a lung specific form.

Sequencing the DNA

• The DNA sequence of the various isoforms reveals their amino acid sequence.

• Once the amino acid sequence is ascertained, the function may be revealed.

Isolating DNA for Sequencing

• E. coli bacteria contain cloned DNA of the isoforms that are being studied.

• In order to retrieve the DNA, the bacterial cells are chemically treated so that they burst. After this, centrifugation and other chemical treatments allow the DNA to be isolated in a pure form for sequencing.

Sequence of isoform 1

Analyzing the Sequence

• Analyzing a DNA sequence involves an array of computing tools– Basic Local Alignment Search Tool (BLAST)

is offered on the NCBI web site to compare sequences to others, which helps determine function relative to that of other sequences.

– Contig express is a software program that allows for the assembly of a finished sequence from fragments

Aligning sequences with AlignX software

• AlignX is a software program that allows a user to compare different sequences.

Comparison of Amino Acid Sequences

Gene Expression Experiments

• Part of answering the “why are there various isoforms” question is to determine where the various isoforms are expressed.

• This can be done by using Real Time Polymerase Chain Reaction

Dime tour of RT-PCR

• PCR uses a template DNA strand, small DNA sequences called primers, and enzymes to produce large quantities of a DNA sequence

• RT-PCR can use dyes to show the progress of an experiment. It can also use enzymes that allow production of DNA from an RNA template

Determining RNA Quality

rRNA Bands

How we use RT-PCR• In order to examine the expression of the various

isoforms, we can use samples of zebrafish mRNA with the DNA primers that we developed based on the sequences of the isoforms.

• The mRNA is first used as a template to produce DNA

• The cDNA samples can be mixed with each of the primers to amplify the amount of DNA. We are able to indicate this with the using fluorescent dyes.

RT-PCR Output

Melt Curve from Expression Experiment

RT-PCR Results

• After conducting the RT-PCR, we noticed that there was significant expression of both isoform 1 and isoform 3 in the gills, muscle, and ovary of the zebrafish.

• We also ran an agarose gel to further examine the amount of DNA produced in this step of the experiment, which backs the results from the RT-PCR reaction

Gel Results from RT-PCR

Conclusions

• Expression of both isoforms is based primarily in Gill and Ovary Tissue.

• Muscle tissue seems to only express isoform 3, as shown in the gel.

• Zebrafish isoform 2 has not been tested; in mammals this isoform is only expressed in lung tissue.

• The gills show the highest expression for both isoforms.

• This is the first report of a third isoform for COX IV in vertebrates.