Does DNA topography coordinate intra- & inter-chromosomal Galactose gene expression? : this thesis is presented in partial fulfilment of the requirements for the degree of Masters of Science in Molecular Biology at Massey University, Albany, New Zealand
For a long time, DNA had been considered as a stabilized, rigid, and “linear” structure,
which acts as a platform for molecular regulators to function. However, genome
structure in living cells is far more complex than the linear representation of the
primary DNA sequence implies. This thesis aims to investigate whether the position
of a gene within the genome plays a role in the regulation of its activity. The galactose
(GAL) gene family of Saccharomyces Cerevisiae is used as a model. This gene family
enables yeast cells to utilize galactose as an alternative carbon source; and it consists
of structural and regulatory genes. Structural genes GAL1, GAL10 and GAL7 exist in
a cluster on yeast chromosome II. The products of the regulatory genes, GAL3, GAL4,
and GAL80, regulate the expression of the GAL structural genes, depending on the
availability of carbon sources. Specifically, GAL gene expression is repressed by
glucose, paused for induction (noninduced) by glycerol/lactate, and fully induced by
The aim of this project was to study the relative position of the GAL structural genes
within the nucleus, and whether any chromosomal interactions at the GAL locus help
to regulate their activation. These were tested in accordance with the expression status
of the GAL genes (i.e. repressed, noninduced or induced). Followed confirmation of
the existence of any chromosomal interactions, protein/protein complexes that
mediate these interactions were attempted to identify.
The methods applied in this project were Chromosome Conformation Capture (3C)
and Circular Chromosome Conformation Capture (4C), which applied in combination
to map the positions of the GAL genes in the context of the overall genome structure.
The results indicated that the GAL locus on chromosome II was divided into two
“interaction zones”. DNA loops formed around these interaction zones to form an
S-shape structure in a carbon source-independent manner. Two novel
inter-chromosomal interactions between chromosomes II and XVI, i.e. SVL3-GAL7
and HOS1-GAL10, were also identified. Although these interactions occurred
regardless of the GAL gene activities, it was suggested by real-time PCR that the
interaction frequency for SVL3-GAL7 declined as the GAL genes being activated.
Unfortunately no protein/protein complexes were identified to play an important role
in mediating either intra- or inter-chromosomal interactions.
Future work will be needed to identify the protein/protein complexes that play a role
in mediating the S-Shape structure at the GAL locus and the two inter-chromosomal
interactions. Additional works could also focus on the understanding of the functional
implication of the interactions between chromosomes II and XVI.