Robert Adams

Summary
The study Divergence in cis-regulatory sequences surrounding the opsin gene arrays of African
cichlid fishes had two main goals: “identify potential cis-regulatory sequences surrounding the
opsin gene arrays of African cichlids (phylogenetic footprinting), and (2) identify those
sequences whose divergence may explain patterns of differential opsin gene expression among
African cichlids (phylogenetic shadowing).” (Kelly E O'Quin, 2011) Cis-regulatory sequences
were chosen as the target of this study because sources of regulatory roles in adaptive
phenotypic evolution are not well understood and because they exhibit codominance and
modularity. The species studied were Oreochromis niloticus and Metriaclima zebra because
they express different wavelength-sensitive opsin palettes as adults and they have a different
evolutionary age. Opsin containing BAC clones for these cichlids were identified through PCR
screening and filter hybridization with subsequent shotgun sequencing using ABI Sanger or 454
Life Sciences technology to perform several analyses. Using MultiPipMaker, non-coding
elements surrounding opsin genes in the O. niloticus BAC and other model cichlids were
highlighted. Looking at areas of high putative conservation among taxa, phylogenetic
footprinting, they identified 20 NCEs. Next, using the Transcription Element Search System,
they used computational predictions to find TFBS within CNEs and proximal promoters and
miRNA target sites within 3’-UTRs. They found 11 locations, within CNEs, of transcription factor
binding sites of 12 known opsins associated transcription factors. Among the many TFBS they
found in proximal promoters, five significantly divergent opsins were identified between M.
zebra and O. niloticus. 2 significantly different 3’-UTRs of opsins between the cichlids were
found when they identified miRNA target sites within those regions. Then they performed
phylogenetic shadowing through comparing the opsin BACs’ and introns’ divergence in the
overall genome to the site divergence previously identified. These comparisons identify the cis-
regulatory sequences that have undergone significant evolutionary divergence. Lastly, they
sequenced the most divergent regions and compared them with other cichlids. Across the one
NCE, two 3’-UTRs, and 5 opsin proximal promoters, they were able to observe a high turnover
rate of CRX TFBS. They conclude they found, “at least nine divergent sequences that could
contribute to opsin expression differences in cis and stand out as candidates for future
functional analyses” (Kelly E O'Quin, 2011).
Background
Nine potential Cis-regulatory sequences surrounding the opsin gene arrays of African cichlids
have been identified because their divergence may explain patterns of differential opsin gene
expression among African cichlids. We believe that cis-regulatory genes account for differences
in African Cichlid gene expression, and, by removing specific CNEs and proximal promoters from
the BAC clones of previously studied cichlids, we will observe a phenotypic change in in adult
Oreochromis niloticus and Metriaclima zebra cichlids with regards to color vision.
To test the potential effects the cis-regulatory genes have on opsin, we must create first
bacteria containing the cichlid opsin DNA, through cDNA amplified by PCR (William D. Comar,
2014). Then mutants must be created missing one opsin each and mutants missing one cis-
regulatory candidate each for a total of 18 strains of mutants, using RNAi. First, we will analyze
our opsin-missing mutants and compare them to the control of cells that have not had any DNA
removed. Using microspectrophotometry (Wolken, 1975) I will be able to observe how much
opsin is in the cells. The amount of opsin needs to be determined when certain genes are
missing to see if the deactivation or removal of potential cis-regulatory genes can deactivate or
cause certain opsins genes to become nonfunctional. Using a spectrophotometer, absorbance
spectra will give us a baseline to evaluate the cis-regulatory mutants against. I expect to see
more absorbance from the unaltered cichlid opsin containing cells, my control for this
experiment, versus the mutants missing one opsin gene. Next, I will test the activity of the opsin
produced by the mutants and control. The mutant strains that are missing an opsin gene are
expected to have no activity recorded on the part of the spectrum that they absorb light. Next,
activity will be recorded and measured using infrared difference spectroscopy. Less activity is
expected to be recorded from the mutants missing certain opsin genes because they will not
have the genes to make the opsin (Siebert, 2001). These two experiments were performed as
controls. It is necessary to know the spectra recordings of cells missing opsins in order to
evaluate whether missing cis-regulatory gene changed an opsins activity or function. Therefore,
we must now test our mutants missing cis-regulatory sequences. The 9 mutants each missing a
cis-regulatory gene needs to be tested for abundance and activity. Microspectrophotometry
will be employed to analyze the abundance (Wolken, 1975) and activity will be observed and
recorded (Siebert, 2001). I expect the cis-regulatory missing mutants will display similar spectra
to that of the mutants’ spectra, whose opsin genes these sequences surround. Analyses will be
very simple. I will perform an anova analyses on each one of my different experiments. I would
get the mean of each mutant and the normal control to compare how their abundance or
activity differed depending on what was missing. I believe that the means of a mutant missing
an opsin gene will correspond to the means of the cis-regulatory genes that surround them.
My hypothesized outcomes would impact the field of adaptive evolution because the exact
mechanism behind rapid change would be known. Humans could turn on and off genes for
genetically heritable pathologies, like cancer. This mechanism could also be employed to help
humans be more suited to their different geographic regions. It would mean we could put the
fast-forward button on developing characteristics that make us more fit. However, it would
raise the question of: how and when do cis-regulatory genes change to cause a phenotypic
alteration.
References
Kelly E O'Quin, D. S.-H. (2011). Divergence in cis-regulatory sequences surrounding the opsin gene arrays
of African cichlid fishes. BMC Evolutionary Biology, 11:120.

Siebert, R. V. (2001). Conformations of the Active and Inactive States of Opsin*. The Journal of Biological
Chemistry, 38487-38493.

William D. Comar, §. S. (2014). Time-Resolved Fluorescence Spectroscopy Measures Clustering and. J.

Am. Chem. Soc., 8342–8349.

Wolken, J. J. (1975). Photoprecesses, Photoreceptors, and Evolution. New York: Academic Press.