Download A Novel Third Isoform of Zebrafish Cytochrome Oxidase IV

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.