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. 2019 Apr 23;116(17):8445-8450.
doi: 10.1073/pnas.1819585116. Epub 2019 Apr 8.

Prediction and Identification of Recurrent Genomic Rearrangements That Generate Chimeric Chromosomes in Saccharomyces cerevisiae

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Free PMC article

Prediction and Identification of Recurrent Genomic Rearrangements That Generate Chimeric Chromosomes in Saccharomyces cerevisiae

Kim Palacios-Flores et al. Proc Natl Acad Sci U S A. .
Free PMC article

Abstract

Genomes are dynamic structures. Different mechanisms participate in the generation of genomic rearrangements. One of them is nonallelic homologous recombination (NAHR). This rearrangement is generated by recombination between pairs of repeated sequences with high identity. We analyzed rearrangements mediated by repeated sequences located in different chromosomes. Such rearrangements generate chimeric chromosomes. Potential rearrangements were predicted by localizing interchromosomal identical repeated sequences along the nuclear genome of the Saccharomyces cerevisiae S288C strain. Rearrangements were identified by a PCR-based experimental strategy. PCR primers are located in the unique regions bordering each repeated region of interest. When the PCR is performed using forward primers from one chromosome and reverse primers from another chromosome, the break point of the chimeric chromosome structure is revealed. In all cases analyzed, the corresponding chimeric structures were found. Furthermore, the nucleotide sequence of chimeric structures was obtained, and the origin of the unique regions bordering the repeated sequence was located in the expected chromosomes, using the perfect-match genomic landscape strategy (PMGL). Several chimeric structures were searched in colonies derived from single cells. All of the structures were found in DNA isolated from each of the colonies. Our findings indicate that interchromosomal rearrangements that generate chimeric chromosomes are recurrent and occur, at a relatively high frequency, in cell populations of S. cerevisiae.

Keywords: PMGL strategy; chimeric chromosomes; genome architecture; genomic rearrangements; reciprocal translocations.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Schematic of the generation and identification of chimeric chromosomes. Two chromosomes sharing an identical repeated sequence are shown. Orange, chromosome A (CHR A); purple, chromosome B (CHR B); gray, repeated sequence; X, centromere. The solid line indicates a region of unique sequences close but outside the repeated region, where the PCR primers must be located; the broken line indicates a fragment of the rest of the chromosomes. Short arrows indicate the relative position of the PCR primers used to detect the wild-type and chimeric structures. Forward primers, a and aI; reverse primers, b and bI. The dotted black line indicates the site where NAHR generated two chimeric chromosomes. If forward primers located in one chromosome and reverse primers located in the other chromosome are used, the chimeric structures are detected. The corresponding structures can be detected either from CHR A–CHR B (Top) or from CHR B–CHR A (Bottom) according to the positions of the forward and reverse primers.
Fig. 2.
Fig. 2.
Location of interchromosomal repeated sequences. Identical repeated sequences larger than 1 kb and shared by different chromosomes were located (Materials and Methods). The chromosomes were arranged as a circle (I–XVI). The position of the centromere is indicated by a black line; the left arm, light gray line; the right arm, dark gray line. Lines joining the corresponding chromosomes indicate the position of each pair of repeated sequences. Orange and green lines join repeated sequences located both either in the left arm or in the right arm of the corresponding chromosomes; green lines indicate the subset analyzed. Purple lines join repeated sequences, one located in the left arm and the other in the right arm of the corresponding chromosomes.
Fig. 3.
Fig. 3.
Genomic location of the PCR fragments derived from wild-type and chimeric structures. Illumina reads from each PCR fragment were obtained (see Materials and Methods). A fragment of the reference genome was cut in the corresponding chromosome (wild-type structure) or chromosomes (chimeric structures) PMGL was used to determine the coverage of the PCR sequence reads along the corresponding reference genome fragment (see Material and Methods). The reference genome fragment is shown as a line bordered by arrows. This fragment is divided into overlapping 25-nucleotide strings shifted by 1 nucleotide. This constitutes the ordered reference string dataset. The rest of the chromosome is shown as a broken line. Each sequence read from the PCR product is similarly divided. This constitutes the PCR read string dataset. Both datasets are merged, and a directed PMGL is generated to indicate the coverage of PCR read strings at each ordered position of the reference genome.
Fig. 4.
Fig. 4.
Origin of the PCR products generated by a wild-type structure involving a transposon. The data were derived from a PCR generated by forward and reverse primers located near but outside the repeated region in chromosome I (CHR I-I). The PCR (ID 2702; SI Appendix, Table S2) shows two products: (A) a large product and (B) a small product (SI Appendix, Fig. S1A). The products are schematized as rectangles. Green, regions upstream and downstream of the transposon (see Results) that correspond to unique sequences in the chromosome; black, regions corresponding to the transposon direct repeated regions or delta sequences; gray, the rest of the transposon or epsilon sequence; line bordered by two-headed arrows, fragment cut from the reference genome; red bars, limits of the large PCR product. The PMGL was generated from the ordered reference string dataset (scale in nucleotides at the top) and the PCR read string dataset from either the large product (PMGL 01_01) or the small product (ID PMGL 027_01) (see SI Appendix, Table S2); coverage of read strings is presented on the y-axis of the corresponding panel. The gray arrow indicates the zone of the transposon that did not attract read strings.
Fig. 5.
Fig. 5.
Origin of the PCR products generated by a chimeric structure involving transposon sequences. The PCR was primed with forward primers located in chromosome II and reverse primers located in chromosome XV. The PCR (ID 2401; see SI Appendix, Table S3) produced a large fragment (A) and a small fragment (B). The products are schematized as rectangles (A, a; B, a) and contain a unique region of chromosome II (orange): the transposon sequences (large fragment) or a fragment of the transposon sequences (small fragment; gray); and a unique region of chromosome XV (purple). Shown in A, b; A, c; B, b; and B, c are the PMGLs obtained using as reference a fragment of the indicated chromosome. Orange arrows, regions that did not attract read strings from chromosome II; purple arrows, regions that did not attract read strings from chromosome XV; gray arrows, regions of the transposon sequences that did not attract read strings from the small fragment. Other indications are as in Fig. 4.
Fig. 6.
Fig. 6.
Estimation of the proportion of chimeric structures relative to wild-type structures. DNA was serially diluted by a factor of 2. Each dilution was used to generate a PCR from either a wild-type structure (here CHR VII, ID PCR 2710; see SI Appendix, Table S2) or a chimeric structure (here CHR I-CHR VII, ID PCR 2382; see SI Appendix, Table S3). Agarose gel electrophoresis was used to detect the corresponding PCR products. (A) wild-type structure; (B) chimeric structure. The first and last lanes of the gels show a reference ladder, with the corresponding sizes in kb shown in the y-axis of each gel. The comparison of the maximal dilution at which chimeric structures are observed with the maximal dilution at which wild-type structures are observed is an indication of the relative number of chimeric structures present in the population. In this case, there are about 23 chimeric structures per ∼217 wild-type structures.
Fig. 7.
Fig. 7.
Identification and estimation of the relative proportion of interchromosomal rearrangements from colonies derived from single cells. The culture of strain S. cerevisiae S288C was plated at high dilution to obtain separated cells. Cells were grown to generate single colonies. DNA was extracted from the culture and from each of four colonies. The proportion of different chimeric structures relative to wild-type structures was estimated as in Fig. 6. Bars indicate the relative number of chimeric structures (y-axis). Black, culture; yellow, colony 1; green, colony2; orange, colony 3; blue, colony 4. Each chimeric structure is indicated at the bottom: the first chromosome is that containing the forward primers; the second chromosome is that containing the reverse primers.

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