Downloaded from genome.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press Letter Recovery of a Function Involving Gene Duplication by Retroposition in Saccharomyces cerevisiae Joseph Schacherer, Yves Tourrette, Jean-Luc Souciet, Serge Potier, and Jacky de Montigny1 Laboratoire de Microbiologie et de Ge´ne´tique FRE 2326, Universite´Louis-Pasteur/CNRS, Strasbourg 67083, France The duplication of DNA sequences contributes to genomic plasticity and is known to be one of the key factors responsible for evolution. The mechanisms underlying these rare events, which have been frequently mentioned by authors performing genomic analysis, have not yet been completely elucidated. These mechanisms were approached here in the yeast Saccharomyces cerevisiae, using a positive selection screen based on a particular mutated allele of the URA2 gene. Spontaneous revertants containing a duplication of the terminal part of the URA2 gene were selected and analyzed. Some important features of the duplicated regions, such as their chromosome location, size, and insertion sites, were characterized. The events selected correspond to a single inter- or intrachromosomal gene duplication process. The duplicated ATCase sequence is generally punctuated by a poly(A) tract and is always located in Ty1 sequences. In addition, the activation of a Ty1 transcription process increased the frequency of the duplication events. All in all, these data suggest that the duplication mechanism involves the reverse transcription of mRNA and the subsequent integration of the cDNA into a Ty1 area. The Ty1 elements and the retrotransposon-encoded function are key factors contributing to chromosomal reshaping. The genomic rearrangements described constitute experimental evidence for the recovery of a function involving duplication by retroposition. Gene duplication can be said to be a key to molecular evolution To understand the mechanisms involved in the duplication (Ohno 1970). It results in redundancy and may be the first step in events at work in S. cerevisiae, we used a genetic screening the emergence of new functions. Duplicated genes may have method developed at our laboratory which can be used for the three types of fate. The most likely of these is a process called positive selection of spontaneous genomic rearrangements nonfunctionalization, in which one of the two copies of a dupli- (Roelants et al. 1995). This screening method is based on the use cate pair may degenerate into a pseudogene or be lost from the of a mutated allele of the S. cerevisiae URA2 gene carrying the genome as the result of the accumulation of several deleterious CPSase and ATCase functional domains (Fig. 1). In the ura2 15- mutations followed by chromosomal rearrangements such as de- 30-72 mutant strain, the ATCase domain is inactive, resulting in letion (Lynch and Conery 2000; Lynch 2002). The second pos- a uracil auxotroph. The functional reactivation of the ATCase sible process, which is called subfunctionalization, consists of a domain suffices to generate Ura+ prototrophs, but only at a low Three types of reactivation events have .10מdivision of the original functions of the ancestral gene between frequency of ∼10 the two duplicates due to the differential accumulation of degen- been described previously: insertion of a Ty1 retrotransposon in erative mutations in both copies (Force et al. 1999). The last and the URA2 coding sequence upstream of the last mutation most important evolutionary gene duplication fate is known as (Roelants et al. 1997), deletion of the sequence bearing the three neofunctionalization. One of the copies in a duplicate pair can mutations (Welcker et al. 2000), and duplication of the ATCase acquire a new function by changing its coding or regulatory se- coding sequence and its insertion elsewhere in the genome under quences, whereas the second copy keeps the original function the dependence of a resident promoter (Bach et al. 1995). (Zhang et al. 1998). Among these three reactivation events, we focused here on Based on experimental studies and data obtained in global the duplication events which lead to the generation of a Ura+ genomic analyses, several hypotheses have been proposed to ex- prototroph. In this case, duplication is synonymous with recov- plain the presence of a duplicated copy. Gene duplication can ery of gene function which is associated with a change in the involve either a single gene, segmental (Bailey et al. 2002; Koszul regulatory sequences involved. We sought to determine the chro- et al. 2004), single chromosome or aneuploidy (Hughes et al. mosome location, size, and insertion sites of the duplicate re- 2000) or whole-genome duplication process (Wolfe and Shields gions in a set of eight spontaneous revertants isolated in a pre- 1997). In Saccharomyces cerevisiae, the only examples of in vivo vious work (Roelants et al. 1995), with a view to establishing the single gene duplication described to date are those involving the underlying gene duplication mechanism. We focused here on ACP1 (Hansche et al. 1978), HIS4 (Greer and Fink 1979), ADH2 four strains in which single ATCase sequence duplication occurs. (Paquin et al. 1992), URA2 (Bach et al. 1995), and hexose trans- In these revertants, the fact that the duplicated segment is punc- port genes (Brown et al. 1998). These duplication events have tuated at the 3Ј end by a poly(A) tract and is located between Ty1 מ מ -dupli- sequences strongly suggests that the Ty1 retrotransposon con 12 10מ been observed at a very low frequency of 10 10 cations per cell per generation, and the mechanisms responsible tributes to this process. To test the role of the Ty1 retrotranspo- for these events are still poorly understood. son in this process, 27 revertants containing a duplication event were selected, starting with a strain in which the Ty1-mediated transposition was enhanced and analyzed: The enhancement of 1Corresponding author. E-MAIL [email protected]; FAX 33 3-90-24-2028. the Ty1 transcription increased the frequency of the genomic Article and publication are at http://www.genome.org/cgi/doi/10.1101/ rearrangements, and that of the duplication events in particular gr.2363004. (Roelants 1996). 14:1291–1297 ©2004 by Cold Spring Harbor Laboratory Press ISSN 1088-9051/04; www.genome.org Genome Research 1291 www.genome.org Downloaded from genome.cshlp.org on September 27, 2021 - Published by Cold Spring Harbor Laboratory Press Schacherer et al. occurred in the four revertants, we estimated the size of the duplicated segment by Southern hybridization using several probes corresponding to regions adja- cent to the URA2 gene. By such a walking procedure, if there is a single gene duplication, only the ATCase probe will show the chromosome carrying the dupli- cation, whereas a segmental duplication process will result in one or several 3Ј adjacent genes of the URA2 locus being coduplicated with the ATCase sequence, and the corresponding probe used on these genes will also show the chromosome carrying the duplicated segment. In the three strains containing interchromo- somal duplications (Rev 25, Rev 37, and Rev 45), the Figure 1 Correspondence between the simplified restriction map of the URA2 locus walking procedure was performed directly on the and protein domains. The thick line indicates the URA2 coding sequence (restriction chromosome by hybridizing the PFGE. The hybridiza- site: b, BglII; B, BamHI). A, C, D, B, and E denote the BamHI-BglII subfragments, ns15 and ns30 the positions of the nonsense mutations, and fs72 the position of the frame- tion of the karyotypes of the strains, using probes spe- shift mutation (GATase, glutamine amidotransferase; CPSase, carbamoylphosphate cific to the YJL131c and YJL132w (MRS3) located at synthetase; DHOase-like, dihydroorotase-like; ATCase, aspartate transcarbamylase). 0.375 kb and 4.16 kb downstream of the URA2 (YJL130w) locus, respectively, yielded only chromo- some X, which indicates that these interchromosomal The genetic screening method based on the URA2 gene pro- duplication events correspond only to the duplication of the AT- vides a powerful tool for understanding the mechanisms in- Case sequence. volved in gene duplication in S. cerevisiae. The results indicate In Rev 56, this procedure could not be used because the that mRNA may mediate spontaneous duplication events and resident and duplicated copies comigrate. In this case, the walk- that Ty elements are involved in the formation of the duplicates. ing procedure was performed on genomic DNA by analyzing the After its integration, the duplicated sequence becomes part of a BamHI patterns with two probes, one corresponding to the chimerical gene characterized by a new pattern of expression. In ATCase coding region and the other to the intergenic region light of these new molecular data, we discuss the possible role of between the ATCase sequence and the YJL131c open reading the Ty1 elements in the chromosomal reshaping process. The frame (ORF). The restriction pattern observed using the ATCase genomic rearrangements described constitute an example of du- probe consisted of two bands, whereas the intergenic probe plication by retroposition. yielded only one band, which indicates that only the ATCase coding sequence was duplicated. RESULTS Characterization and Chromosome Location Sequence Analysis of the Insertion Sites of the Duplication Events of the Duplicated Regions Starting with an initial ura2 15-30-72 mutant strain, four spon- The insertion sites of the