Copyright Ó 2005 by the Genetics Society of America DOI: 10.1534/genetics.105.047670 Monitoring the Mode and Tempo of Concerted Evolution in the Drosophila melanogaster rDNA Locus Karin Tetzlaff Averbeck and Thomas H. Eickbush1 Department of Biology, University of Rochester, Rochester, New York 14627-0211 Manuscript received July 1, 2005 Accepted for publication August 25, 2005 ABSTRACT Non-LTR retrotransposons R1 and R2 have persisted in rRNA gene loci (rDNA) since the origin of arthropods despite their continued elimination by the recombinational mechanisms of concerted evolution. This study evaluated the short-term evolutionary dynamics of the rDNA locus by measuring the divergence among replicate Drosophila melanogaster lines after 400 generations. The total number of rDNA units on the X chromosome of each line varied from 140 to 310, while the fraction of units inserted with R1 and R2 retrotransposons ranged from 37 to 65%. This level of variation is comparable to that found in natural population surveys. Variation in locus size and retrotransposon load was correlated with large changes in the number of uninserted and R1-inserted units, yet the numbers of R2-inserted units were relatively unchanged. Intergenic spacer (IGS) region length variants were also used to evaluate changes in the rDNA loci. All IGS length variants present in the lines showed significant increases and decreases of copy number. These studies, combined with previous data following specific R1 and R2 insertions in these lines, help to define the type and distribution, both within the locus and within the individual units, of recombinational events that give rise to the concerted evolution of the rDNA locus. ANDEMLY repeated multigene families frequently ensuring that all ribosomal subunits are equally com- T undergo concerted evolution, a phenomenon in patible with other components of the translational which genes in a gene family show more sequence ho- machinery. On the basis of population genetic studies, mogeneity within a species than between species. It has various recombinational models for explaining the been suggested that homogenization occurs most rapidly homogeneity of the tandemly repeated rRNA genes within a chromosome (Schlo¨tterer and Tautz 1994) have been proposed (Coen et al. 1982; Lyckegaard and by recombinational mechanisms such as gene conver- Clark 1991; Schlo¨tterer and Tautz 1994; Polanco sion, intrachromosomal loop deletions, and unequal et al. 1998, 2000). crossovers between sister chromatids (Dover 1994; Elder An added complexity to understanding the evolution and Turner 1995; Liao 1999). New sequence variants of the rDNA loci is that in many animal phyla these loci within an array can also increase in frequency and are home to specialized transposable elements (Eickbush spread through a population by segregation and recom- 2002; Burke et al. 2003; Kojima and Fujiwara 2004; bination between homologs and can eventually become Penton and Crease 2004). Best studied are the R1 and fixed in the species by natural selection, molecular R2 non-LTR retrotransposable elements of arthropods. drive, or drift. These elements insert into the 28S gene and render the The ribosomal RNA gene locus (rDNA) is of partic- inserted genes nonfunctional (Long and Dawid 1979; ular interest for the study of concerted evolution. In Kidd and Glover 1981; Eickbush and Eickbush eukaryotes, the rDNA locus is composed of hundreds to 2003). R1 and R2 have persisted via vertical descent in thousands of tandemly repeated rRNA genes inter- arthropods since the origin of the phylum, suggesting spersed with noncoding, intergenic spacer (IGS) regions that occasional retrotransposition has been an effective (Long and Dawid 1980). High redundancy of rRNA strategy to evade elimination from the rDNA locus by genes is critical for fitness because the ribosomal trans- the recombinational mechanisms of concerted evolu- lational machinery of the cell is necessary in large quan- tion (Burke et al. 1998; Malik et al. 1999; Gentile et al. tities for growth and the RNA components of the 2001). ribosome structure do not benefit from translational To derive a comprehensive population genetics model amplification. Homogenization of the repeats within for the evolution of the rDNA locus and its R1 and R2 species is thought to be beneficial to the organism by inhabitants, one must measure changes in multiple pro- perties of the locus over time. These properties include 1Corresponding author: University of Rochester, Hutchison Hall, the number of rDNA units, the sequence of the units, Rochester, NY 14627-0211. E-mail: [email protected] IGS length variation, the fraction of the units inserted Genetics 171: 1837–1846 (December 2005) 1838 K. T. Averbeck and T. H. Eickbush with R1 and R2, the frequency of R1 and R2 retrotrans- R2 elements, and the primer 59-TGCCCAGTGCTCTGAAT position, and, finally, the distribution of this variation GTC-39, which anneals to the 28S gene sequence 60–80 bp across the locus. To acquire these data, we are conduct- upstream of the R2 insertion site. End labeling of the R2 primer, PCR amplification of both male and female genomic DNA, ing a long-term study of the rDNA loci in the Harwich and separation of the PCR products on 8% high-voltage dena- mutation-accumulation lines of Drosophila melanogaster. turing polyacrylamide gels are described in Pe´rez-Gonza´lez The Harwich lines are replicate stocks derived from a and Eickbush (2002). PCR amplifications of 59 ends of full- highly inbred line, separated over 400 generations ago length R2 elements produced multiple distinct product e´rez onza´lez ickbush (Mackay et al. 1992). Using the highly variable 59 ends lengths (see P -G and E 2002, Figure 3A). The relative intensity of each band was quantitated from the of R1 and R2 that are generated during insertion, the PhosphorImager scan. To adjust for the increased PCR am- rates of R1 and R2 retrotransposition and elimination plification efficiency of shorter DNA fragments, the expected in these lines have been previously estimated (Pe´rez- intensity per copy was calculated with a regression analysis Gonza´lez and Eickbush 2002; Pe´rez-Gonza´lez et al. using single copy variants as reference markers. PCR amplifi- 2003). This report continues our characterization of the cation of the full-length R2 elements in males produced 11–16 different PCR fragments, representing variants in both the evolutionary dynamics of the Harwich rDNA loci, this X- and the Y-linked rDNA locus. Within each amplification time with an emphasis on locus structure. We quanti- reaction, bands were defined as representing single copy or tated differences in the X-chromosome-linked rDNA multicopy variants. The copy numbers of more intense, multi- loci, including the number of units in each locus, the copy bands were then determined. Female DNA in all stocks load of transposable element insertions, and variation produced either four or five fragments (X-linked locus only). Comparison of the bands generated from male and female in the IGS. Our results show a remarkably dynamic locus DNA from each line was used to confirm which of the X-linked with significant changes in its size and composition in bands represented single copies and to provide data for the only 400 generations. The combined analysis of R1, R2, linear regression. and IGS markers in these lines provides insights into the properties of the recombinational mechanisms that RESULTS drive the concerted evolution of the rDNA locus. Variation in the fraction of 28S genes containing R1 and R2: The fraction of X-chromosome-linked 28S MATERIALS AND METHODS rRNA genes inserted with R1 and R2 was determined for each Harwich line by quantitative Southern analysis Fly stocks and DNA isolation: The Harwich stocks were a of genomic DNA. This approach was possible because of gift from T. F. C. Mackay. Line number designations were con- sistent with line numbers in Mackay et al. (1992), and flies the high level of sequence uniformity of the 28S rRNA were collected at various times from the 395th to the 415th gene, the R1 and R2 elements, and the 39 junction of generation. Genomic DNA was isolated from 50 females and these elements with the 28S gene (Eickbush and 75 males per line as described in Eickbush and Eickbush Eickbush 1995; Lathe et al. 1995; Lathe and Eickbush (1995). 1997). To conduct the analysis, Southern blots of triple Southern genomic blots: For the genomic blots, 3 mg DNA was restriction digested and separated on 1.0% agarose gels. restriction digested (ClaI, BamHI, and PstI) female DNA After transfer of the genomic DNA to nitrocellulose paper, the from each stock was probed with a 28S gene fragment paper was hybridized in 23 SSC, 53 Denhardt’s at 65° for located downstream of the R1 and R2 insertion sites. 14 hr as described in Eickbush and Eickbush (1995). Final A diagram of the 28S gene indicating the locations of washing of the filters was in 0.53 SSC at 65°. Gene sequences restriction sites and the probe used in the analysis is used for the hybridization probes were amplified via PCR from genomic DNA. PCR primers 59-TTAGTGGGAGATATTAGA shown in Figure 1A, and an example of a resulting geno- CCTC-39 and 59-TGAACACCGAGATCAAGTC-39, which am- mic blot is shown in Figure 1B. The restriction digest plified a region extending from position 6100 to 6521 (Tautz produced three fragment types: uninserted 28S genes et al. 1988), were used to generate the 28S probe, and (2.3-kb ClaI-ClaI), 28S genes inserted with R2 (1.5-kb 59-GCCGACCTCGCATTGTTC-39 and 59-TTTGTATTATACC PstI-ClaI), and 28S genes inserted with R1 (0.7-kb GTAACG-39, which amplified a region extending from posi- tion 10881 to 11184 (Tautzet al. 1988), were used to generate BamHI-ClaI). The rare 28S genes with both R1 and R2 the external transcribed spacer (ETS) probe.