Expression of tandem gene duplicates is often greater than twofold

Abstract

Tandem gene duplication is an important mutational process in evolutionary adaptation and human disease. Hypothetically, two tandem gene copies should produce twice the output of a single gene, but this expectation has not been rigorously investigated. Here, we show that tandem duplication often results in more than double the gene activity. A naturally occurring tandem duplication of the Alcohol dehydrogenase (Adh) gene exhibits 2.6-fold greater expression than the single-copy gene in transgenic Drosophila This tandem duplication also exhibits greater activity than two copies of the gene in trans, demonstrating that it is the tandem arrangement and not copy number that is the cause of overactivity. We also show that tandem duplication of an unrelated synthetic reporter gene is overactive (2.3- to 5.1-fold) at all sites in the genome that we tested, suggesting that overactivity could be a general property of tandem gene duplicates. Overactivity occurs at the level of RNA transcription, and therefore tandem duplicate overactivity appears to be a previously unidentified form of position effect. The increment of surplus gene expression observed is comparable to many regulatory mutations fixed in nature and, if typical of other genomes, would shape the fate of tandem duplicates in evolution.

Keywords: gene expression; gene structure; genome evolution; position effect; tandem duplication.

Fig. 1.

Tandem duplication of Adh from D. virilis is overactive. (A) ADH enzyme activity is sixfold higher in D. virilis than D. americana. Boxplots show median and interquartile range with thin lines extending to the lesser of 1.5× the interquartile range or the data extremes. n = 15 samples. (B) Schematic of the tandem duplicated Adh locus in D. virilis (“Dup”). Vertical bars delimit the duplicated region. Ovals mark the three nucleotides that distinguish the left copy from the right copy. Also shown are engineered constructs with SNPs removed (“Ident_dup”) and the isolated single copy (“Single”). (C) ADH activity of D. melanogaster flies (ZH-86Fb attP site, Adh-null) transformed with D. virilis Single and Dup constructs. Dashed line shows predicted twofold mean activity of the Single construct. Error bars show 95% confidence interval of means (Tables S1–S14). Sample sizes for this and subsequent plots are in Tables S1–S14. We verified that assay measurements scaled one-to-one with homogenate concentration (Fig. S1).

Fig. 2.

Excessive ADH activity is due to the tandem duplication, not copy number per cell. F2 homozygotes and heterozygotes from crosses back to the Adh-null transgene insertion line were extracted using the high-throughput procedure. Compare the Single homozygote and the Dup heterozygote, each of which bear two copies of the Adh but in different configurations.

Fig. 3.

Duplicate overactivity is not limited to Adh and varies with genomic position. vgQ-lacZ “Single” and tandem duplicate (“Dup”) constructs were inserted in the following attP sites: (A) ZH-86Fb (same site and genetic background as Fig. 1); (B) ZH-22A; (C) ZH-68E; (D) ZH-51D; (E) VK00037; (F) VK00033; and (G) attp40 (same site and genetic background as Fig. 4). β-Galactosidase activity was measured from wing imaginal discs. Dashed line shows predicted twofold activity of Single construct at each site. (H) Summary of preceding panels. Each point represents mean β-galactosidase activity from Dup and Single inserts at each site. Dashed line indicates a twofold activity difference.

Fig. 4.

Tandem duplicate activity is simply additive for Adh in one genomic position. (A) ADH activity for virilis Adh Single and Dup constructs in the attP40 site in Adh-null background. (B) Differences in ADH activity are proportional to copy number per cell. F2 heterozygous and homozygous males from crosses back to the Adh-null transgene insertion line were generated as in Fig. 2.

Fig. 5.

Overactivity is associated with increased transcription. Expression of Adh relative to control gene RP49 was measured with quantitative real-time PCR. (A) Adh transcription from duplicates in the ZH-86Fb site is overactive. (B) Adh transcription from duplicates in the attp40 site is additive. Dashed line shows predicted twofold expression of Single construct.

Fig. S1.

Standard curves. To ensure linearity of assay conditions, standard curves were constructed. Regression equations and R² values for linear regressions on log₂-transformed values are shown on each chart. (A) ADH activity of twofold serial dilution of fly homogenates. Circles: Ident_dup flies. Triangles: Single construct flies. Open symbols denote data points that we excluded from linear regression due to loss of linear response at low concentrations. Arrowhead marks the fly concentration used in enzyme assay experiments. (B) β-Galactosidase activity of twofold serial dilution of purified β-galactosidase enzyme under assay conditions. Arrowheads mark the range of activity values observed from wing imaginal discs in experiments. (C and D) qRT-PCR of fourfold serial dilution of virilis Adh (C) or RP49 (D) plasmids. Arrowheads mark the range of quantification cycle (C_q) values observed from fly cDNA in experiments.

Fig. S2.

Comparison of homogenization methods. F2 homozygotes and heterozygotes from crosses to the null pf86 line give qualitatively similar results when extracted using (A) Potter–Elvehjem homogenizers or (B) the miniG ball bearing grinder (same data as Fig. 2).