The document summarizes an experiment that studied the effects of natural selection on core promoter regions (CPRs) of gene regulatory regions in Drosophila melanogaster. Researchers obtained sequence and transcriptome data from 29 lines of D. melanogaster from the Drosophila Genetic Reference Panel. They analyzed single nucleotide polymorphisms, gene expression levels, and linkage between CPRs and surrounding regions. The results showed that nucleotide changes in CPRs caused gene expression variation and were influenced by both positive and balancing selection. Sex-biased gene expression was also common but CPRs were not dependent on sex-specific expression. In conclusion, CPRs are a source of natural selection and gene expression variation within populations of D. melanogaster.

1. Jason Gramling
Professor Neil Blackstone
Bios-317 Evolution
June 21, 2016
Variation in Gene Regulatory Regions
The article that I chose describes an experiment that uses of Drosophila melanogaster to study
the effects of natural selection and variance on core promotor regions of gene regulatory
regions. The fly’s’ genome is from a natural population in North Carolina that is stored in the
Drosophila Genetic Reference Panel (DGRP), a community resource for analysis of population
genomics. This data from the DFRP is easily available and was used in the experiment to test
the natural selection. The article describes that studies have shown the adaptive evolution is
related to the evolution of gene regulatory sequences (Carroll, 2005). Variations in gene
expression are considered to affect phenotypic consequences in morphology, physiology,
behavior, and disease susceptibility. Sequence variations in regulatory regions are thought to
be crucial for phenotypic variation.
Variation can be detected by a few methods such as nucleotide diversity and Tajimas D test and
were used in this experiment. Procedures involving transcriptomic technologies, including
“microarray and high-throughput RNA sequencing,” allows scientists to observe variation in

2. gene expression in natural populations of species, including human kind. Scientists can detect
sequence variations in gene regulatory regions that cause gene expression variation that has
been subject to natural selection.
Core Promoter Regions or CPRs are genetic regions that direct the start of transcription by RNA
polymerase II and contain several sequences that interact with other proteins associated with
the start of transcription Scientists know more about these CPRs than most other complex
regions of the genomic structure of eukaryotes.
In the experiment, several core promoter regions were detected as candidates for the test. One
of them was CHKov1, which provides the fly with resistance to certain viruses and insecticides.
29 lines of sequence data for D. melanogaster were obtained from the DGRP, and Single
nucleotide polymorphisms (SNPs) were also gathered from the database. Additionally,
Transcriptome data was also extracted and used in the experiment. Where Transcriptome data
is a collection of all transcribed gene data of a genome (National Human Genome Reserach
Insitute, 2015).
This data was ran through several tests including DNA microarray, where DNA fragments are
attached to a surface and then analyzed or scanned (Stranger, et al., 2005). To test for Natural
selection the scientists conducting the study created a model of the fly population in North
Carolina. A statistical test called Tajima's D was used 100,000 times to determine balancing and
positive selection. Linkage between CPRs and surround regions was also tested via haplotype
blocks and haploview.

3. Overall the experiment concluded that nucleotide changes in CPRs caused variation and
affected the expression of genes. Of the 11,454 known CPRs, 6799 were expressed with high
broad-sense heritability. The average length for the CPRs was 169.4 base pairs while the
median of the regions was 160 base pairs. Also, changes in CPRs were increased by positive
selection, but maintained by balancing selection, or basically the changes were selected against
and brought the number of individuals with the changes back down. This maybe so due to the
expression of a gene giving the fly a resistance to viruses and insecticides, while the changes in
CPRs could also cause issues with flies motor functions and behavior. The experiment also
revealed that CPR sequences varied greatly even within a population and possibly providing a
source of Natural selection. The results also showed that sexual dimorphism for gene
expression is a common pattern due to almost 80% of the expressed transcripts was influenced
greatly by sex biased gene expression. Sex biased genes create and sustain expressed
differences between male and females (Assis, Zhou, & Bachtrog, 2012). However the study
found that CPRs are not dependent on sex-specific expression, or genes being expressed in only
one sex and not the other.
Brief summaries of the 3 cited research papers:
Evolution at Two Levels: On Genes and Form (Carroll, 2005)
The journal article covers a broad research into regulatory genes and proteins. It starts off to
describe a research paper done 30 years ago comparing human and chimps anatomical and
behavioral differences, which concluded that the level of differences between them could not
just be explained by the small level of molecular differences between the two species. The

4. author explains that since then, there have been many changes in what we know to what
actually causes the differences. Since then scientists have found since then how regulatory
genes and their proteins act upon expression of genes. Additionally, it was also found that gene
structure and function regulation plays a factor in species differentiation. More recently, it has
been found that changes in gene regulation can affect the gain, loss or the modification of
organisms’ traits. Also that it can be many regulatory sites could affect many different
expressions of the genes for the traits.
The journal also gives an examples of these type of changes in regulatory genes and their
affects. For instance, Hox genes in insects; these genes are involved in the development of
embryos. Different linages of insects have changes in the number of Hox genes, complete loss
of the genes, while even others loss the Hox genes and then gained other functions. All of these
changes expressed many different results and species.
The article concluded that main differences between chimps and man are not because of small
molecular differences like what the 30 year old article concluded, but by the differences in gene
regulatory regions and gene expressions.
The paper reviews the recent advances in human knowledge of how changes in gene regulatory
areas of genomes affect changes in species and their resulting mutations. The article covers
multiple families, and their genes and proteins. It is much boarded and general than the main
article concerning natural selection of gene regulatory regions. The main article however does
build on the ideas of this paper where it specifies and narrows down the generalization to just

5. one species. The original paper does reinforce the theories of how changes in gene regulatory
regions by showing that those regions are affected by natural selection on that level.
Neutral and adaptive variation in gene expression (Whitehead & Crawford, 2006)
This study aims to identify the relation of gene expression variation evolving by natural
selection and random-neutral changes in populations. The experiment involves measuring the
expression of genes that are involved with a fish, Fundulus heteroclitus metabolism. This animal
was selected due to its natural habitat range having a drastic change in temperature and its
ability to adapt to said temperature differences. The fish were collected from several points
along the US east coast from the Atlantic Ocean. The article points out that there have been
studies like this one done before, but only for single genes, while this study will include an array
of genes. 329 genes involved with metabolism were investigated with the experiment. They
were measured by multiple methods and found that 44 genes were being acted upon by
natural selection among the sample of fish collected.
This experiment detailed in this reference paper focuses on finding out if and what genes are
being acted upon by natural selection in a population of fish. It is very similar to the original
paper where both used arrays of genes and identified what forms of natural selection was
being used. The original paper reinforces the main idea from this paper that natural selection
does act on genes. It also provides another example of a different species that is affected by
this idea. The main difference is that the original paper involves gene regulatory regions, while
the reference article involves gene expression.

6. Gene expression variation in African and European populations of Drosophila melanogaster
(Hutter, Saminadin-Peter, Stephan, & Parsch, 2008)
The article details an experiment that compared gene expression variation in 16
D. melanogaster strains from Africa and Europe. The strains were not laboratory strains but
pulled from natural populations, with 8 from each locale. It was also the largest survey of the D.
melanogaster at that time. The whole genome of the fruit fly was compared via microarrays to
study gene expression variation between the two different populations. From the study it was
found that the two strains did not vary much between the two populations. However there was
a much higher of gene expressions within the populations themselves. It was also found that X-
linked genes also had less expression changes that autosomal genes in both populations.
The reference article does show on how D. melanogaster can differ on a gene expression level.
This species of course was used in the original papers experiment with the difference of that
the natural habitat for the original paper was North Carolina, USA. Like the original paper it
shares the same results, with the differences between individuals within single population
greater than compared to a different population. This might have impacted the selection of the
flies gathered for the original papers experiment. As trying to compare two separate regions
population might lead to little variation between the as it did in the reference paper
experiment.

7. References
Assis, R., Zhou, Q., & Bachtrog, D. (2012). Sex-biased transcriptome evolution in Drosophila.
Genome Biology and Evolution, 1189-1200.
Carroll, S. B. (2005). Evolution at Two Levels: On Genes and Form. PLOS Biology, 245.
Hutter, S., Saminadin-Peter, S. S., Stephan, W., & Parsch, J. (2008). Gene expression variation in
African and European populations of Drosophila melanogaster. Genome Biology, R12.
National Human Genome Reserach Insitute. (2015, 8 27). Transcriptome. Retrieved from
National Human Genome Reserach Insitute:
https://www.genome.gov/13014330/transcriptome-fact-sheet/
Sato, M. P., Makino, T., & & Kawata, M. (2016). Natural selection in a population of Drosophila
melanogaster explained by changes in gene expression caused by sequence variation in
core promoter regions. BMC Evolutionary Biology, 16-35.
Stranger, B., Forrest, M., Clark, A., Minichiello, M., Deutsch, S., & al, e. (2005). Genome-wide
associations of gene expression variation in humans. PLoS Genet, 78.
Whitehead, A., & Crawford, D. L. (2006). Neutral and adaptive variation in gene expression.
Proceedings of the National Academy of Sciences of the United States of America, 5425-
5430.

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