Intragenic tandem repeats generate functional variability

Abstract

Tandemly repeated DNA sequences are highly dynamic components of genomes. Most repeats are in intergenic regions, but some are in coding sequences or pseudogenes. In humans, expansion of intragenic triplet repeats is associated with various diseases, including Huntington chorea and fragile X syndrome. The persistence of intragenic repeats in genomes suggests that there is a compensating benefit. Here we show that in the genome of Saccharomyces cerevisiae, most genes containing intragenic repeats encode cell-wall proteins. The repeats trigger frequent recombination events in the gene or between the gene and a pseudogene, causing expansion and contraction in the gene size. This size variation creates quantitative alterations in phenotypes (e.g., adhesion, flocculation or biofilm formation). We propose that variation in intragenic repeat number provides the functional diversity of cell surface antigens that, in fungi and other pathogens, allows rapid adaptation to the environment and elusion of the host immune system.

Fig. 1. S. cerevisiae genes containing conserved intragenic repeats

A screen of all open reading frames in the S. cerevisiae genome for those containing conserved intragenic tandem repeats identified 29 genes with large (≥ 40 nt) repeats (panel a) and 15 genes with short (<40 nt) repeats (panel b). Repeats (vertical boxes) that vary in size among 6 different S. cerevisiae strains are colored red (see text and Supplementary Fig. 1-2 online); repeats that do not show size variation among these strains are colored green. Cell surface genes are indicated in blue. The numbers in panel b indicate the number of repeats. More information about the repeats (consensus sequence, conservation…) is given in Supplementary Table 1 online. The repeat units in most genes are distinct from those in others except in FLO1, FLO5 and FLO9, which share the same repeat unit, and in PIR1, PIR3 and HSP150, which share the same repeat unit. SED1, FIT1, SLA1 and MSB2 contain two intragenic repeat regions with different repeat sequences. YOL155C and DAN4 contain three distinct repeat regions.

Fig. 2. Intragenic repetitive domains vary in size

The repetitive domains of all 44 genes carrying intragenic repeats (Fig. 1) and the ORFs of 16 control genes without repeats were amplified by PCR for six different S. cerevisiae strains. This figure shows the results for 5 genes with repeats (FLO1, FLO11, PIR3, NUM1 and CSH5) and one gene without repeats (DIA3). Lane 1: S288C (haploid); Lane 2: Sigma1278b (haploid); Lane 3: EM93 (diploid); Lane 4: CMBS355 (polyploid); Lane 5: CMBS DL16 (polyploid); Lane 6: CMBS33 (polyploid). The analysis of other genes is in Supplementary Figures 1-3 online. The variability in some cell surface genes has already been used for the genotyping of wine yeasts.

Fig 3. Intragenic repeats are hot-spots for recombination

(a). In order to monitor recombination between intragenic repeats in the FLO1 gene, a URA3 expression cassette was integrated at various positions in the FLO1 repeats. As a consequence of the numerous recombination events within the repeats, the URA3 marker is lost at exceptionally high frequencies, resulting in a 5-FOA-resistant (Ura⁻) strain containing a new FLO1 allele. (b). Assay for loss of the URA3 marker. Ura⁺ strains (KV315, URA3 integrated at its native locus in the genome) grow on minimal medium (SC -Ura), but not on 5 fluoro-orotic acid (5-FOA) medium. Ura⁻ strains (BY4742) grow on 5-FOA but not on minimal medium. Strains KV132 and KV133, with a URA3 cassette in the FLO1 repeats (FLO1::URA3), grow on minimal medium. Due to recombination events within the repeats, the URA3 cassette is looped out at high frequencies, yielding many 5-FOA-resistant segregants. (c). S. cerevisiae strains with the URA3 cassette integrated into various positions in the genomic FLO1 repeats (FLO1::URA3) were grown on medium lacking uracil and subsequently plated onto 5-FOA medium. The numbers indicate the frequency of Ura⁻ segregants.

Fig 4. Repeats in pseudogenes provide an additional source of variability

(a). Most of the recombination events between FLO1 repeats are strictly intragenic, i.e. only repeats within FLO1 recombine with each other. However, in some cases, the repeats in FLO1 recombine with a similar repeat unit found in the FLO1 pseudogene YAR062W, which is located about 12 kb downstream of FLO1. Fusion of a repeat in FLO1 with that in YAR062W results in deletion of the 3′ end of FLO1 and the entire 12 kb of DNA separating both open reading frames. (b). The FLO1-YAR062W deletion/fusion results in altered mobility of chromosome I (231 kbp) using clamped homogeneous electrical field (CHEF) electrophoresis. Lane 1: wild-type S. cerevisiae S288C; Lanes 2: control Ura⁻ segregant (KV291) that has lost only intragenic FLO1 repeats. Lanes 3-4: FLO1-YAR062W fusion strains (KV360 and KV361). (c). Southern analysis confirms the deletion of the 12 kb region in between FLO1 and YAR062W. Genomic DNA of wild-type cells (lane 1) and the FLO1-YAR062W fusion strains (lanes 2-3) was cut with Pst1 and used for Southern blotting with probes that bind to the 5′ portion of FLO1 (probe 1, top) and the 3′ portion of FLO1 (probe 2, bottom). Other probes were used to further confirm the fusion (not shown).

Fig. 5. Instability of the FLO1 repeats generates functional variability

S. cerevisiae strain KV133 (FLO1::URA3) was plated onto 5-FOA medium to select Ura⁻ segregants. (a) The Ura⁻ segregants harbor FLO1 alleles of different lengths, ranging from 2.9 kb to 5.4 kb. (b) PCR amplification of the FLO1 repetitive domains shows that the different lengths of the alleles are due to corresponding differences in the lengths of the FLO1 repeat region. Lane 1: strain KV298 (FLO1 ORF = 2.9 kb); lane 2: KV308 (3.1 kb), lane 3: KV220 (3.7 kb), lane 4: KV219 (4.1 kb), lane 5: KV224 (4.5 kb), lane 6: KV211 (4.6 kb), lane 7: KV312 (5.0 kb), lane 8: KV311 (5.4 kb). (c) Expansion of FLO1 repeats leads to increased adherence to polystyrene. The FLO1 genes of the S288C strain BY4742 (KV210, lane C1) and of 8 strains containing different-sized FLO1 alleles (see panel a) were fused to the GAL1 promoter. Cells were grown on galactose medium and subsequently tested for adhesion to polystyrene by staining with crystal violet. Cells expressing a long allele of FLO1 showed strong adhesion to polystyrene, whereas cells expressing shorter alleles do not adhere. When grown on glucose, strain KV311 fails to adhere (lane C2). Strain KV306, which contains the same FLO1 allele as strain KV311, but lacks the GAL1 promoter, fails to adhere when grown on galactose medium (lane C3). (d). Linear relationship between adherence to polystyrene and number of repeats. The error bars represent the standard deviation between three independent experiments. (e). Expansion of FLO1 repeats results in stronger cell-cell adhesion. The pGAL1-FLO1 fusion strains (tubes 1-8, see panel b) were tested for flocculation. Cells expressing a long allele of FLO1 show extremely strong cell-cell adhesion (all cells have sedimented on the bottom of the test tube). Tube C contains a strain (KV210) carrying the wild-type FLO1 allele of S. cerevisiae strain BY4742 fused to the GAL1 promoter.