Construction and validation of yeast artificial chromosome contig maps by RecA-assisted restriction endonuclease cleavage

Abstract

RecA-assisted restriction endonuclease (RARE) cleavage is an "Achilles' heel" approach to restriction mapping whereby a RecA-protein-oligodeoxynucleotide complex protects an individual restriction site from methylation, thus limiting subsequent digestion to a single, predetermined site. We have used RARE cleavage to cut yeast artificial chromosomes (YACs) at specific EcoRI sites located within or adjacent to sequence-tagged sites (STSs). Each cleavage reaction produces two YAC fragments whose sizes are a direct measure of the position of the STS in the YAC. In this fashion, we have positioned 45 STSs within a contig of 19 independent YACs and constructed a detailed RARE-cleavage map that represents 8.4 Mbp of human chromosome 6p21.3-22. By comparing maps of overlapping YACs, we were able to detect seven internal deletions that ranged from approximately 75 kbp to approximately 1 Mbp in size. Thirteen pairs of EcoRI sites were targeted for double RARE cleavage in uncloned total human DNA. The excised fragments, up to 2 Mbp in size, were resolved by pulsed-field gel electrophoresis and were detected by hybridization. In general, the genomic RARE-cleavage results support the YAC-based map. In one case, the distance in uncloned DNA between the two terminal EcoRI sites of a YAC insert was approximately 1 Mbp larger than the YAC itself, indicating a major deletion. The general concept of RARE-cleavage mapping as well as its applications and limitations are discussed.

Figure 1

RARE-cleavage-mapping strategy. (A) Determining the extent of overlap between two YACs. The right end of the insert cloned in YAC 2 is isolated, sequenced, and tagged with an STS (○). An oligodeoxynucleotide (□) containing the end sequence up to and including the EcoRI site at the vector-insert junction (E2) is used to split the overlapping clone YAC 1 at E2. The size of the left RARE-cleavage fragment is a direct measure of the extent of overlap. The clone overlap can also be measured by RARE cleavage of YAC 2 at E1. (B) Generating and placing a microsatellite marker. A plasmid-clone library is prepared from EcoRI-digested YAC-1 DNA and is screened by hybridization with a CA-repeat probe. Once a suitable marker has been found (•), the sequence information necessary for designing a RARE-cleavage oligonucleotide at one of the flanking EcoRI sites (■) can be obtained by simply sequencing the end of the plasmid insert the marker came from. RARE cleavage of YAC 1 at this EcoRI site (E3) produces two fragments whose sizes indicate the position of the microsatellite marker within the YAC. (C) Clone validation. Uncloned genomic DNA is subjected to double RARE cleavage by using two oligonucleotides for the two EcoRI sites defining the ends of the YAC-1 insert at once. The resulting fragment should have the same size as the EcoRI fragment cloned in YAC 1.

Figure 2

Mapping of a YAC by RARE cleavage at 15 EcoRI and 2 AluI sites. (A) YAC y950h11 was subjected to RARE cleavage at 15 EcoRI sites. Two reactions were set up for each site containing 40 μg of RecA protein and either 1.65 μg (left lane of each pair) or 0.83 μg (right lane) of oligonucleotide. The mapping landmarks associated with the RARE-cleavage sites are listed along the top. The relative positions of the cleavage sites within the YAC are illustrated on the right. The two arrows denote the inverted duplication that includes D6S2243 and D6S2242. (B) RARE cleavage and control experiments were performed on YAC y950h11 by using either the EcoRI (lanes 1–5) or the AluI restriction/modification system (lanes 6–12). Samples in lanes 1 and 6 were only treated with methylase. Lanes 2 and 7 contain DNA that was completely digested with restriction endonuclease. Samples in lanes 3 and 8 were first methylated and then incubated in the presence of restriction enzyme. Samples in lanes 4 and 5 and 9–12 were complete RARE-cleavage reactions containing 40 μg of RecA protein and 1.32 μg (lanes 4, 9, and 11) or 0.66 μg (lanes 5, 10, and 12) of oligonucleotide. Lanes 4, 5, 9, and 10 contain RARE-cleavage reactions for D6S1558. Samples cleaved at an AluI site near y899 g1-L were loaded in lanes 11 and 12. PFG electrophoresis was performed at 6 V/cm by using a switching time ramped from 60 to 120 s. DNA molecules containing only the left (A) or either YAC-vector arm (B) were detected by hybridization. The positions of selected lambda-concatemer and H. wingei size markers are indicated.

Figure 3

Comparison of RARE-cleavage fragments from two overlapping YACs. Two yeast colonies containing YAC y901a10 (lanes 1–3 and 10–12) were analyzed by RARE cleavage at the EcoRI site defining the right end of YAC y935a8. Lanes 4–9 contain samples from two different DNA preparations of YAC y935a8 that were cleaved at the EcoRI site at the right end of YAC 901a10. The leftmost lane within each group of three contained undigested DNA. The cleavage reactions contained 40 μg RecA protein and either 1.32 μg (middle) or 0.66 μg (right lanes) of oligonucleotide. The products were separated on a PFG, which was run for 24 h at 6 V/cm, and a switching time ramped from 35 to 70 s. The hybridization probe was a mixture of pBR322 and λ DNA to visualize the λ-concatemer size markers. The positions of YACs and of RARE-cleavage fragments are indicated on the right. y935a8 is 2.3 Mbp in size. Both full length and deleted y935a8 as well as the right-vector-arm containing fragments thereof migrate in the unresolved limiting-mobility band. The right end-fragment of the deleted y901a10 clone (DEL-y901a10-R; lanes 2 and 3) is smaller than the right end-fragment from the intact version (y901a10-R; lanes 11 and 12), which in turn has the same size as the left end-fragment from y935a8 (y935a8-L; lanes 8 and 9). DEL-y935a8-L is a deleted fragment that was observed in one particular y935a8 preparation. The hybridization signal at the bottom of lanes 11 and 12 is due to a spill-over from the λ ladder.

Figure 4

Test of YAC integrity by RARE cleavage of uncloned human DNA. Total human DNA was cut by RARE cleavage at two pairs of EcoRI sites that define the ends of two YACs. (A) Lanes 1–3 contain total human DNA that has been digested with SfiI (lane 1) or NotI (lane 2) or that has been first methylated with EcoRI methylase and then treated with both NotI and EcoRI restriction endonucleases (lane 3). The RARE-cleavage reactions (lanes 4 and 5) contained 80 μg of RecA protein and 1.32 μg or 0.66 μg of each of the two oligonucleotides specific for the insert ends of y950h11. Lane 6 contains y950h11 DNA. The PFG was run for 28 h at 6 V/cm with a reorientation angle of 120° and 75- to 150-s switching times. (B) Lane 1 contains NotI-digested total human DNA. The sample in lane 2 was first methylated with EcoRI methylase and subsequently exposed to both EcoRI and NotI restriction endonucleases. The RARE-cleavage reactions (lanes 3–6) contained 80 μg of RecA protein and 2.64, 1.98, 1.32, or 0.66 μg of each of the two oligonucleotides directed at the terminal EcoRI sites of y947f6. Lane 7 contains y947f6 DNA. The gel was run for 60 h at 3 V/cm, 106° with 300–600 s switching time. Each gel was blotted to a nylon membrane that was cut between the human and yeast-DNA containing lanes. Fragments produced by digestion of human DNA were detected by hybridization with the JFp19 (A) or the cDNA 25 probe (B). Autoradiography was for 2 d. The complete digests serve as positive controls for the sensitivity of hybridization and degree of methylation (20). The multiple bands in the NotI digest in B may be due to partial digestion, partial methylation, or cross-hybridization. The 3.13-Mbp size marker in B runs at the limiting mobility where the high local DNA concentration may cause unspecific hybridization. The yield of RARE-cleavage product decreases at lower oligonucleotide concentrations, and no product band is visible at the lowest concentration (lane 5 in A, lane 6 in B). This effect is due to unspecific binding to DNA of excess RecA protein leading to incomplete methylation and unspecific cleavage. The YAC-containing filter strips were probed with pBR322 and were exposed for less than 5 h to match the intensity of the bands in lanes containing human DNA.

Figure 5

RARE-cleavage map representing 8.4 Mbp of human chromosome 6p21.3–22. YACs are represented as horizontal lines whose lengths are proportional to their size. Thin lines indicate noncontiguous end pieces of chimeric clones or YAC ends whose chromosomal origin is unknown. L and R refer to the left (centric) and right (acentric) YAC-vector arm, respectively. Vertical ticks mark the positions of RARE-cleavage sites within each YAC. Maps of individual YACs were aligned graphically. A consensus map indicating the coordinates of 47 cleavage sites and the inferred positions of two clone ends for which no RARE-cleavage assay has been developed (∗) is shown at the top. The solid dots mark RARE-cleavage assays that require AluI. The bracket at ≈4 Mbp denotes the inverted repeat structure mentioned in the text. Bold horizontal lines are map segments whose apparent length is consistent, within experimental error, between at least two independent clones. Triangles indicate length inconsistencies that were interpreted as internal deletions with the approximate extent of the deletion indicated in kilobase pairs. The absence of HLA-F in YAC y960h11 (ellipse) is known from its STS content; the extent of this deletion is not known. Other missing ticks simply mean that these assays have not been performed on a particular YAC, except for the “X” at 2.5 Mbp in y669h7 where cleavage attempts have failed consistently. Open bars represent fragments generated by double RARE cleavage of uncloned total human DNA along with their respective hybridization probes with the approximate fragment sizes indicated in megabase pairs. Only the size of the largest fragment is given for sets of nested fragments (drawn as “stacks”).