Rare germinal unequal crossing-over leading to recombinant gene formation and gene duplication in Arabidopsis thaliana

Abstract

Small, multigene families organized in a tandem array can facilitate the rapid evolution of the gene cluster by a process of meiotic unequal crossing-over. To study this process in a multicellular organism, we created a synthetic RBCSB gene cluster in Arabidopsis thaliana and used this to measure directly the frequency of meiotic, intergenic unequal crossing-over between sister chromatids. The synthetic RBCSB gene cluster was composed of a silent ⌬RBCS1B::LUC chimeric gene fusion, lacking all 5 transcription and translation signals, followed by RBCS2B and RBC3B genomic DNA. Expression of luciferase activity (luc ؉ ) required a homologous recombination event between the ⌬RBCS1B::LUC and the RBCS3B genes, yielding a novel recombinant RBCS3B͞ 1B::LUC chimeric gene whose expression was driven by RBCS3B 5 transcription and translation signals. Using sensitive, single-photon-imaging equipment, three luc ؉ seedlings were identified in more than 1 million F2 seedlings derived from self-fertilized F1 plants hemizygous for the synthetic RBCSB gene cluster. The F2 luc ؉ seedlings were isolated, and molecular and genetic analysis indicated that the luc ؉ trait was caused by the formation of a recombinant chimeric RBCS3B͞1B::LUC gene. A predicted duplication of the RBCS2B gene also was present. The recombination resolution break points mapped adjacent to a region of intron I at which a disjunction in sequence similarity between RBCS1B and RBCS3B occurs; this provided evidence supporting models of gene cluster evolution by exon-shuff ling processes. In contrast to most measures of meiotic unequal crossing-over that require the deletion of a gene in a gene cluster, these results directly measured the frequency of meiotic unequal crossingover (Ϸ3 ؋ 10 ؊6 ), leading to the expansion of the gene cluster and the formation of a novel recombinant gene.Genome organization can directly affect the evolution of a gene. Single-copy genes or dispersed members of a multigene family evolve independently. In contrast, members of a multigene family organized as a gene cluster can exploit this organization to generate further gene duplications and novel recombinant genes by a process of unequal crossing-over. For example, a single intergenic unequal crossover event in a gene cluster results in four simultaneous alterations: a deletion, a duplication, and two reciprocal, recombinant genes. The impact of such unequal crossover events evidently have been important in the evolution of complex loci such as HOX (1), amylase (2), globin (3), MHC (4), Ig (5), the maize R-r complex (6), RBCS (7), and plant disease-resistance loci (8-10). Although DNA sequencing of gene clusters provides information about past changes in a particular gene cluster, it can only estimate the rate of unequal crossing-over in terms of geological time scales.In multicellular organisms, unequal crossing-over is implicated in several genetic disorders. This was demonstrated first with the Drosophila bar locus (11). Subsequent research with the bobbed locus demonstrated ...

The effects of trans modifiers and tandem duplications on meiotic recombination in maize

“…Similarly, at a synthetic tandemly arrayed gene cluster in Arabidopsis, meiotic unequal interchromatid recombination events tended to resolve in regions of higher sequence identity between the duplicated sequences JELESKO et al 1999). In yeast, rates of meiotic recombination were significantly reduced by increased sequence divergence between inverted repeat sequences (CHEN and JINKS-ROBERTSON 1999).…”

“…Similar to homologous recombination between single-copy sequences, unequal interhomolog and interchromatid recombination between duplicated sequences ( Figure 2) has been observed in yeast (JACKSON and FINK 1981;JACKSON and FINK 1985;KLEIN 1984;KLEIN and PETES 1981;MOTOVALI-BASHI et al 2004;SZOSTAK and Wu 1980;THOMPSON and STAHL 1999), tobacco (TOVAR and LICHTENSTEIN 1992) and Arabidopsis (ASSAAD and SIGNER 1992;JELESKO et al 1999;MOLINIER et al 2004;OPPERMAN et al 2004). Also similar to equal recombination, unequal interchromatid recombination predominated during mitosis in a study conducted in yeast (JACKSON and FINK 1981), whereas unequal interhomolog recombination was preferred in meiosis (JACKSON and FINK 1985).…”

“…Even so, the findings that 1) rates of unequal recombination at Al-b were higher in the presence of a homolog than in its absence (i.e., hemizygotes) and 2) recombinants isolated from marked hétérozygotes most often showed an exchange of flanking phenotypic markers (LAUGHNAN 1949;LAUGHNAN 1952), were interpreted to suggest that the homolog is the preferred recombination template (LAUGHNAN 1952). More recently, molecular characterizations in plants have documented both meiotic interhomolog (ASSAAD and SIGNER 1992;MOLINIER et al 2004;TOVAR and LICHTENSTEIN 1992) and interchromatid (ASSAAD and SIGNER 1992;JELESKO et al 1999;MOLINIER et al 2004;TOVAR and LICHTENSTEIN 1992) unequal recombination events. Meiotic unequal recombination events isolated from synthetic direct repeats in Arabidopsis (MOLINIER et al 2004) and synthetic inverted repeats in tobacco (TOVAR and LICHTENSTEIN 1992) were conducted using both homozygotes (in which recombination can occur either between homo logs or sister chromatids) and hemizygotes (in which recombination can occur only between sister chromatids).…”

“…Few unequal recombination studies have included molecular characterization of both interhomolog and interchromatid recombinants in plants ( JELESKO et al 1999;MOLINIER et al 2004). Genetic studies in maize have contributed to our knowledge of unequal recombination at tandemly duplicated genes.…”

“…Unequal recombination breakpoints (n=25) were distributed non-randomly in recombinants isolated from hemizygotes (i.e., unequal recombination between sister chromatids) in a synthetic tandemly arrayed cluster of RBCSB genes JELESKO et al 1999).…”