A test of the coordinated expression hypothesis for the origin and maintenance of the GAL cluster in yeast

Abstract

Metabolic gene clusters--functionally related and physically clustered genes--are a common feature of some eukaryotic genomes. Two hypotheses have been advanced to explain the origin and maintenance of metabolic gene clusters: coordinated gene expression and genetic linkage. Here we test the hypothesis that selection for coordinated gene expression underlies the clustering of GAL genes in the yeast genome. We find that, although clustering coordinates the expression of GAL1 and GAL10, disrupting the GAL cluster does not impair fitness, suggesting that other mechanisms, such as genetic linkage, drive the origin and maintenance metabolic gene clusters.

Figure 1. The GAL1-GAL10-GAL7 gene cluster in Saccharomyces cerevisiae.

(A) GAL1, GAL10, and GAL7 encode enzymes that catalyze sequential steps in the assimilation of galactose. Gal1 is the galactokinase. Gal10 contains two catalytic domains: a mutarotase that interconverts galactose enantiomers, and an epimerase domain that converts UDP-galactose to UDP-glucose. Gal7 is the galactose-1-p uridyl transferase. An intermediate in galactose assimilation, galactose-1-p, is toxic to cells. (B) GAL1, GAL10, and GAL7 are clustered within a 7 kb region on Chromosome II with GAL1 and GAL10 sharing a divergent promoter.

Figure 2. To determine the effect of gene clustering on the coordinated expression of the GAL genes, we monitored production of Gal1-GFP and Gal10-mCherry (or Gal7-mCherry) in both the cis and trans conformations following a 2.5 g/L galactose pulse into a steady-state glucose-limited (0.8 g/L) chemostat.

GFP and mCherry were quantified by flow cytometry. (A) Population profiles showing the correlation (R²) between Gal1-GFP and Gal10-mCherry (or Gal7-mCherry) following the galactose pulse. (B) Correlation coefficients (R²) between Gal1-GFP and Gal10-mCherry as a function of time following the galactose pulse. Gal1-GFP and Gal10-mCherry are more correlated in the cis conformation (0.82 at 200 min) compared to the trans conformation (0.63 at 200 min). For Gal1-GFP and Gal7-mCherry, however, we find no difference in the coordination of gene expression between the two conformations (0.70 and 0.69 for cis and trans, respectively at 200 min). The correlation between Gal1 and Gal7 in either conformation is similar to Gal1-Gal10 in the trans conformation. (C) The average cell density (± one standard deviation) for all eight populations following the galactose pulse as measured by Coulter counter. Although the Gal proteins were detectable 30 minutes, cell number did not increase until 120 minutes subsequent to the galactose pulse.

Figure 3. Strategy for disrupting the contiguity of the GAL cluster starting from the prototrophic S288c strains DBY12000 (MATa) and DBY12001 (MATα).

Construction of strains hemizygous for each of the three GAL-cluster genes required three rounds of transformation replacing GAL7, GAL10, and GAL1 with HphMX, KanMX, and NatMX, respectively. Prior to mating, the haploid strains were backcrossed to DBY12000 (or DBY12001) carrying either GFP or dTomato in order to fluorescently label strains for the competition experiment. Note that each of the four possible hemizygous was constructed twice independently, and are indicated by open and closed circles in Figure 4.

Figure 4. To determine if GAL gene clustering provides a selective advantage, we generated GFP-labeled strains hemizygous for GAL1, GAL10 and GAL7, in either the cis conformation, or with one of the GAL genes in trans ( Figure 3 ) and measured their fitness by competing each against a dTomato-labeled GAL cis strain in three conditions: glucose (YPD), galactose (YPG), and alternating glucose and galactose.

Open and filled circles represent independently constructed biological replicates of the hemizygous strains (Figure 3). In glucose there is no difference in fitness for any of the hemizygous strains. The homozygous GAL (wild-type) strain has a 0.9% advantage and the homozygous galΔ strain has a 0.8% fitness disadvantage compared to the hemizygous reference strain. Since the GAL genes are repressed in glucose, this fitness difference is due to the presence of the three drug cassettes (KanMX, NatMX, and HphMX) absent from the wild-type strain, present in one copy in the reference strain, and in two copies in the galΔ strain. In galactose, the galΔ strain is quickly outcompeted, however all other strains have a slight advantage over the reference strain, suggesting a slight advantage to GFP over dTomato in galactose (0.7±0.3%). The lack of a fitness difference between wild-type and the hemizygous strains in galactose suggests two things: (1) the cost of the drug markers in mitigated in galactose (perhaps because of slower growth in galactose and/or because the GAL genes are overexpressed under this condition and may incur a cost themselves), and (2) the hemizygotes are able to maintain adequate levels of the Gal proteins despite a two-fold reduction in gene copy number. Under alternating conditions, the wild-type strain has a 4.3% advantage indicating that increased copy number of the GAL genes, which does not affect fitness when growing exclusively in galactose, is beneficial in a changing environment, likely by establishing steady state levels of the Gal proteins more rapidly. In the alternating regime, like in galactose, disrupting the contiguity of the GAL1-GAL10-GAL7 cluster does not impair fitness; GAL10-trans and GAL7-trans may, in fact, have a fitness advantage.