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Link to original content: https://pubmed.ncbi.nlm.nih.gov/10468603
Rare germinal unequal crossing-over leading to recombinant gene formation and gene duplication in Arabidopsis thaliana - PubMed Skip to main page content
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. 1999 Aug 31;96(18):10302-7.
doi: 10.1073/pnas.96.18.10302.

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

Affiliations

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

J G Jelesko et al. Proc Natl Acad Sci U S A. .

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 DeltaRBCS1B::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 DeltaRBCS1B::LUC and the RBCS3B genes, yielding a novel recombinant RBCS3B/ 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 RBCS3B/1B::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-shuffling 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 crossing-over (approximately 3 x 10(-6)), leading to the expansion of the gene cluster and the formation of a novel recombinant gene.

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Figures

Figure 1
Figure 1
Genetic constructs and isolation of luc+ seedlings. (a) The A. thaliana RBCSB locus. The black line indicates noncoding genomic DNA. Exons shown in color: RBCS1B (green), RBCS2B (orange), and RBCS3B (red). Sizes of introns and exons are not to scale but rather represent genetic organization. Restriction enzymes: P, PflMI; B, BsmI; and S, SphI. Labeled black half-arrows indicate respective oligonucleotide primer-binding sites. (b) Synthetic RBCSB gene cluster construct. NPTII gene is shown in gray. ΔRBCS1BLUC fusion consisted of RBCS1B sequences from the PflMI in exon I to the BsmI site in exon III; firefly luciferase-NOS 3′ terminator (blue) was cloned in-frame 3′ to RBCS1B exon III. RBCS2B-RBCS3B sequences were positioned 3′ to the ΔRBCS1BLUC fusion. (c) An unequal crossover event between sister chromatids containing the synthetic RBCSB gene cluster. Red boxes with black borders define the region of gDNA amplified by PCR with the respective oligonucleotide primers. (d) Ten-minute photon-counting image of empty imaging chamber. (Inset) Pseudocolor step gradient depicting low photon density (light blue) to high photon density (red). [Bar = 4 cm (d–g).] (e) Ten-minute photon-counting image of a tray containing approximately 7,500 F2 seedlings with 1 luc+ seedling (red spot). (f) Image from e superimposed on reflected green-light image of the same tray on which toothpicks were placed to approximate the location of the luc+ seedling. (g) Twenty-five seedlings transferred from the tray in f onto a 0.8% water agar Petri plate. A photon-counting image superimposed on a reflected green-light image allowed the unambiguous identification of a single luc+ seedling on the Petri plate.
Figure 2
Figure 2
RBCS3B/1BLUC responsible for luc+ trait. (a) Genomic DNA was isolated from plants and subjected to PCR by using o13 (RBCSB promoter-recognition) and o14 (LUC-specific) oligonucleotide primers. PCRs were separated on a 3.5% nondenaturing acrylamide gel. MW, molecular weight markers; Col-0, untransformed wild type; 203.10, luc transgenic line containing the synthetic RBCSB gene cluster used to make F1 and F2 generations; 204.7, luc+ transgenic positive control line AtJGJ204.7; 4A1, 6F1, and 6G1, luc+ F2 isolates; pJGJ204, plasmid DNA used to make transgenic positive control line AtJGJ204.7. (b) Genomic DNA was cut with SphI and subjected to DNA blot analysis by using a 32P-dCTP-labeled LUC-NOS probe. DNA was loaded in each lane as indicated: Col-0, wild-type Col-0; Ler, wild-type Landsberg erecta; 203.10, luc transgenic line containing synthetic RBCSB gene cluster used to make the F1 population; 203.15, independent luc transgenic line containing a synthetic RBCSB gene cluster; 204.7, luc+ positive control line AtJGJ204.7; 4A1, 6F1, and 6G1, luc+ F2 isolates. (c) PCR amplification products from genomic DNA using an oligonucleotide primer pair specific for RBCS2B-3B intergenic region (o37) and LUC (o22) (see Fig. 1) were separated on a 0.8% agarose gel.
Figure 3
Figure 3
5′ DNA sequence of the chimeric RBCS3B/1BLUC genes. 4A1, 6F1, 6G1, and AtJGJ204.7 PCR fragments shown in Fig. 2a were sequenced, and 600 bp of 5′ DNA sequence were aligned with genomic RBCS1B and RBCS3B DNA sequences. Underline indicates the o13 oligonucleotide sequence used in the PCR. Bases shown in bold type indicate RBCS3B-specific signatures. Box indicates translation initiation codon in exon I. Arrow indicates PflMI restriction site used to make the 5′ boundary of the ΔRBCS1BLUC chimeric gene. Vertical lines define intron–exon boundaries. Shaded areas define the regions in which the recombination resolution break point must have occurred.

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