Evolution of gene expression and gene-regulatory sequences in Drosophila melanogaster

In this work, I investigate the role of gene regulatory changes in
the evolution of Drosophila melanogaster. As a first step, I
performed a survey of gene expression variation in the species
using whole-genome microarrays. I surveyed eight strains from an
ancestral African population and eight strains from a derived
European population using an experimental design that allowed for
the detection of expression differences within and between
populations. Levels of gene expression variation were nearly equal
within the two populations, but a higher amount of variation was
detected in comparisons between the two populations. Most gene
expression variation within populations appears to be limited by
stabilizing selection. However, some genes that are differentially
expressed between the two populations might be targets of positive
selection. Some of these encode proteins associated with
insecticide resistance, food choice, lipid metabolism, and flight.
These genes are good candidates for studying adaptive regulatory
evolution that accompanied the out-of-Africa migration of D.
melanogaster. To verify the accuracy of the microarray experiments,
I performed quantitative Real-Time PCR (qPCR), which is another
method to measure gene expression, on a subset of genes. I compared
the fold-changes in gene expression between pairs of strains
determined by the two methods. I also compared the pattern of
expression variation in male and female flies. The qPCR approach
supported the general accuracy of the microarray experiments, as
the fold-changes measured by the two techniques were highly
correlated. Expression differences among the strains tended to be
similar for male and females. However, exceptions to this general
pattern could be found by looking at the pairwise fold-changes for
individual genes, some of which differed in expression pattern
between males and females. I also investigated the molecular
evolution and population genetics of the protein-encoding and
upstream regulatory regions of genes that have potentially
undergone adaptive evolution at the gene-regulatory level. These
genes represent a subset of the genes that showed a significant
difference in gene expression between the African and European
populations. A set of control genes, which showed no significant
difference in expression between the two populations, was also
included in the analysis. Overall, I found evidence for both
positive and purifying selection in the coding and non-coding
regions. However, patterns of polymorphism and divergence did not
differ significantly between the candidate genes and the control
genes. One of the genes that showed an interesting pattern of
expression in the microarray and qPCR experiments was subjected to
further, more detailed population genetic analysis. This gene,
CG9509, has twofold higher expression in the European strains than
in the African strains. The coding and the upstream regions of this
gene show evidence of recurrent positive selection since the split
of D. melanogaster and its close relative, D. sechellia. A
polymorphism survey of the CG9509 region uncovered a 1.2-kb
segment, which included the putative CG9509 promoter that showed no
polymorphism in the European population. The European population
also has several fixed or nearly-fixed derived mutations in this
region. These observations, coupled with statistical analysis,
provide evidence for a selective sweep in the European population.
The selective sweep was likely driven by local adaptation at the
level of gene expression.