Optimum conditions for selective isolation of genes from complex genomes by transformation‐associated recombination cloning

Sun-Hee Leem 1 2 Vladimir N. Noskov 2 Jung-Eun Park 1 2 Seung Il Kim 0 Vladimir Larionov 2 Natalay Kouprina 2 0 Proteome Analysis Team, Korea Basic Science Institute , Daejon 305-806, Korea 1 Department of Biology, Dong-A University , Pusan 604-714, Korea 2 Laboratory of Biosystems and Cancer, Center for Cancer Research, National Cancer Institute, National Institutes of Health , Bethesda, MD 20892, USA Transformation-associated recombination (TAR) cloning in yeast is used to isolate a desired chromosomal region or gene from a complex genome without construction of a genomic library. The technique involves homologous recombination during yeast spheroplast transformation between genomic DNA and a TAR vector containing short 5 and 3 gene-specific targeting hooks. Efficient gene capture requires a high yield of transformants, and we demonstrate here that the transformant yield increases ~10-fold when the genomic DNA is sheared to 100-200 kb before being presented to the spheroplasts. Here we determine the most effective concentration of genomic DNA, and also show that the targeted sequences recombine much more efficiently with the vector's targeting hooks when they are located at the ends of the genomic DNA fragment. We demonstrate that the yield of gene-positive clones increases ~20-fold after endonuclease digestion of genomic DNA, which caused double strand breaks near the targeted sequences. These findings have led to a greatly improved protocol. - We recently developed a recombinational cloning strategy, named transformation-associated recombination (TAR) cloning, to isolate large genomic fragments (1,2). The method exploits a high level of recombination between homologous DNA sequences during transformation in the yeast Saccharomyces cerevisiae. For isolation, genomic DNA is transfected into yeast spheroplasts along with a TAR vector that contains targeting hooks homologous to the genomic DNA. Recombination between the vector DNA and the genomic DNA fragments results in establishment of yeast artificial chromosomes (YACs). Propagation of TAR-generated YACs in yeast cells depends on acquisition of genomic DNA with ARS-like sequences that can function as origin-of-replication sites in yeast. These short, degenerative, AT-rich sequences are distributed throughout all eukaryotic genomes, averaging one ARS per 2040 kb (3,4). Thus, most eukaryotic chromosomal regions can be isolated by TAR cloning as YACs, with the insert size ranging from 50 to 250 kb. When common repeats (such as SINEs and LINEs) are inserted into the vector as targeting hooks, TAR cloning produces genomic libraries in which each insert is flanked with a common repeat. A TAR approach has been applied successfully to the construction of genomic libraries for several organisms, including human (1,2) and mouse (5). The method has also been used to clone human DNA selectively from monochromosomal humanrodent hybrids and radiation hybrids containing chromosome fragments using a vector containing human-specific Alu repeats (1,2,68). It is worth noting that for some genomes, cloning in yeast may be the only way to construct large insert libraries. Dictyostelium discoideum DNA, for example, because of its high AT content and inverted repeats, is unclonable in bacterial artificial chromosome (BAC) vectors (9). In addition, TAR cloning provides a unique opportunity to selectively isolate a chromosomal segment of interest or a gene from complex genomes using vectors with unique targeting hooks developed from 5 and 3 sequences flanking the segment. We have been using this method in our laboratory for the past few years and have isolated dozens of human and mouse single-copy genes and specific chromosomal fragments (1018). Because TAR cloning produces multiple gene isolates, it allows the isolation of both parental alleles of the gene which can then be used for haplotype analysis (19). The technique also provides an opportunity to clone gene homologs and mutant genes from clinical material (20). Now that the draft sequencing phase of the Human Genome Project (HGP) has been completed, TAR cloning can be applied to other aspects of the project, including closing the gaps and verifying contig assembly. It may enable the isolation and characterization of gap sequences that cannot be propagated as BACs in Escherichia coli cells while they are stable as YACs in yeast cells (21). Another potential application of the method to the HGP is for closing the biggest gaps in the human genomethe centromeric regions. These regions generally have been excluded as HGP targets because of the difficulty of cloning large blocks of repetitive DNAs (22). We recently used TAR cloning to isolate clones of centromeric DNA up to ~300 kb in length with high selectivity (23). TAR cloning has become a routine and valuable procedure in our laboratory. Our continued investigation of conditions that maximize the efficiency and selectivity of gene capture, and their description here, will make the procedure available to the rest of the scientific community. Specifically, we determined the smallest homologous region required for gene isolation (4), and we developed a system allowing selection of gene-positive clones genetically (24). To generalize the technique, we developed a novel TAR cloning strategy that can isolate genomic regions regardless of the presence of ARS-like sequences (V.N.Noskov, N.Kouprina, S.H.Leem, I.Ouspenski, J.C.Barrett and V.Larionov, manuscript submitted); that approach allows isolation of genomic regions not only from eukaryotes, but also from prokaryotes. Still, not all TAR cloning conditions are optimized, and that makes it difficult for other laboratories to adopt the technique for routine use. In the present study, we investigated various aspects of the technique and described what works best for us. We found the quantity and size of genomic DNA that yields the greatest number of transformants, and we demonstrated that double strand breaks (DSBs) adjacent to the targeted sequence in genomic DNA increase the yield of gene-positive clones dramatically. MATERIALS AND METHODS Yeast strains and transformation The highly transformable S.cerevisiae strain VL6-48N (MATa, his3-D1, trp1-D1, ura3-D1, lys2, ade2-101, met14 ciro), which has HIS3 and URA3 deletions, was used for the transformation experiments (20). Spheroplasts were prepared as described previously (24). Cell density of the culture of yeast used to prepare spheroplasts is critical, and ~2 3 107 cells/ml is optimal in our laboratory. We count cells with a hemacytometer rather than estimate their number by optical density because the OD660 of a culture varies from ~1.2 to 4.5, depending on the type of spectrophotometer. Yeast transformants were selected on synthetic complete medium plates lacking histidine (16). When an ARS-containing TAR vector was used for cloning, transformants were selected on synthetic his plates containing 1 mg/ml of 5-fluoro-orotate (5 (...truncated)