Human Genome Cloning And Isolation Of Specific Dnas In Y

Environmental Health Sciences

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Abstract

Opportunities to manipulate natural and artificial chromosomes have greatly enhanced the utility of S. cerevisiae for addressing functional genomics issues. We developed an in vivo approach for site-directed mutagenesis, based on transformation with oligonucleotides. The cloning-free system, referred to as delitto perfetto provides for the rapid in vivo creation of products having only the desired mutation in a target of approximately 200 bp. We showed that single and multiple base substitutions, insertions and deletions of 1 to 16000 bp, can be accomplished with high efficiency and accuracy. The approach relies on the integration and successive precise excision of a COunterselectable-REporter (CORE) cassette and exploits the proficient homologous recombination system of yeast. Transformation by oligonucleotides that have homology to small regions around the CORE provide for targeted replacement of the core. By varying the sequence of the oligos, precise mutations including substitutions and small and large deletions can be accomplished. Oligonucleotide targeting is completely dependent on the function of RAD52 and it is 15-25 fold less efficient in rad50, mre11 and mre11 nuclease deficient (D16A) backgrounds. A set of four CORE cassettes was created to make the system applicable for all haploid strains, including wild type non-auxotrophic strains. In addition to the common heterologous genes URA3Kl, G418 and Hygromycin resistance, we developed a novel counterselectable marker based on the human p53 allele V122A (Inga et al., Oncogene, 2001). The altered transactivation specificity of this allele prevents growth when overexpressed, without affecting genome stability. While the technique has many applications and the underlying mechanisms are interesting for studies of recombination and genome rearrangments, there have been limitations. For example, the delitto perfetto approach would greatly benefit if it were independent of genetic background, could produce large genome rearrangements, and could be used to create modifications in essential genes, even in genes that affect homologous recombination. This led to the investigation of a double-strand break (DSB) mediated process. The delitto perfetto-DSB system is a modification of the delitto perfetto mehotd that provides a dramatic improvement with an over 1000-fold increase in efficiency, resulting in considerably greater versatility and high throughput generation of genetic alterations. The delitto perfetto-DSB approach, exploits the use of induced DSBs in the genome to increase oligonucleotide targeted mutagenesis. A DSB-CORE cassette is inserted by standard DNA targeting procedures at a desired site. The insertion site is anywhere in the sequence which has been chosen to be deleted or is close to a site where a specific mutation is to be created. The DSB-CORE cassette contains the following: i). Gal1/10 promoter fused to an I-SceI open reading frame (Gal10/1::I-SceI). Any inducible (i.e., on/off) promoter can be used and any DSB site-specific cutting enzyme, provided that the only DNA site cut in the cell is located in the cassette. ii) I-SceI cut site (or other unique cut site that is the target of a DSB site-specific cutting enzyme) located at one-end of the cassette or internally. iii) COunterselectable REporter genes (i.e., the counterselectable gene Kl-URA3 + the kanMX4 reporter gene). Immediately prior to transformation, cells are grown in the presence of galactose, which induces I-SceI endonuclease expression. The enzyme targets its single site in the cassette and generates a DSB. Transformation of cells with oligonucleotides leads to loss of the cassette, creation of the desired mutation(s), or deletion of the desired region. Oligonucleotide targeting in the vicinity of the DSB (delitto perfetto-DSB method) is increased up to 1000-fold, compared to oligo-mediated changes without the DSB (initial delitto perfetto method). The delitto perfetto-DSB has greatly expanded opportunities for gene/chromosome modification, particularly since it can be accomplished in cells with an inactive recombination system. This approach system is also being used to study basic mechanisms of double-strand break repair.