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Undergraduate Research at UMass Dartmouth
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will be present in both the symbiotic clownfish
and non-symbiotic closely related species but is
inactive in the skin of symbiotic species. My goal
was to test primers on tissues of anemonefish and
closely related non-symbiotic species to see where
expression occurred.
To accomplish these goals, I would:
• Use degenerate PCR to obtain partial sequences
of SIAT7 from symbiotic and non-symbiotic
fish species.
• Use inverse PCR to determine the sequences of
the regions surrounding the SIAT7 gene.
• Use quantitative PCR to determine which tissues
express SIAT7 in symbiotic and non-symbiotic
fish species.
To approach this, I knew that SIAT7 had been identified
in close relatives of clownfish. If primers were
designed based from those sequences and added
to DNA of symbiotic clownfish, there would be a
product formed if the primers found complementary
parts of the DNA. In the spring, I completed
degenerate PCR to try to acquire partial sequences
of SIAT7 from non-symbiotic fish species. The
degenerative primers were created from the bicolor
damselfish (Stegastes partitus; Genbank accession
XP_008298796.1), and PCR was done on cDNA samples
from the ocellaris clownfish (Amphiprion ocellaris)
and the non-symbiotic Springer’s damselfish
(Chrysiptera springeri) which is a close relative. The
PCR yielded some products that are bright bands
in the gel below (Figure 1.). The brighter the bands
the more concentration of DNA, showing successful
replication. These samples were then cleaned
up and sent off for sequencing. The sequencing
results were crosschecked with the NCBI database
and the matches that appeared were not of SIAT7.
Instead they matched to other genes like protein
FAM20A isoform X3 inform the Southern pig-tailed
macaque or monkey (Macaca nemestrina) (Figure
2.) or to bacterial genes like protein A2680_02525
from the bacteria Candidatus kaiserbacteria. These
sequencing results are the DNA of one of the bands
from the failed attempt using degenerate PCR.
These results indicate that our DNA in the degenerate
PCR was not successful at producing a partial
part of the SIAT7 gene.
Since the degenerate PCR primer was not successful
at yielding a partial sequence for SIAT7, another
approach to obtaining this sequence was taken.
Marian Wahl, a graduate student in Dr. Robert Drew’s
lab, had recently sequenced transcriptomes from
several species of anemonefish and non-symbiotic
fish. Transcriptomes are all of the RNA that is
made by genes of an organism. This is of interest
because it shows exactly what is made and what will
potentially be translated to proteins. This was not
available in the spring but became available early
this summer. I redesigned primers for four species
of anemonefish (list species) and non-symbiotic fish
(list species) to be used in the PCR. This provided me
with a better chance of getting PCR product because
the primers used in the PCR were designed from the
exact species they would be testing in. Also, I would
be able to see right away if SIAT7 was really present
in the fish species because I would be checking