Gene Disruptiondeletion In Saccharomyces Onestep Gene Replacement

Gene disruption is a method by which a DNA fragment is used to replace a genome sequence with a selectable marker gene, such as HIS3 or kanavanine resistance. In so doing, a deletion is created. The process occurs by homologous recombination and uses the enzymes of the homologous recombination pathway, such as Rad52p. The ends of the exchange fragment must be long enough and have sufficient homology to the chromosomal site so that homologous recombination can occur. Moreover, the size of the region to be deleted can be quite large but must be contained in a single chromosome.

One-step gene replacement is a relatively efficient process. Free DNA ends are very 'recombinogenic' in yeast. This means that free 3' and 5' ends of double-stranded DNA fragments in vivo search out homologous sequences in the chromosomes with very high efficiency. When a homologous sequence is found, the free ends invade the chromosomal sequence and this leads to a crossover event at a site near the free end. If this happens at both ends of a DNA fragment, then the fragment replaces the genomic copy. This is shown in Figure 1.3 for a fictitious gene, YGI1 (Your Gene of Interest).

Recombinant DNA methods can used to construct the disruption fragment. This method was used prior to the development of polymerase chain reaction (PCR)-based methods (see below) and is often seen in the literature. Disruption constructs for many genes are available from researchers in the yeast community and are provided upon request. To make a disruption construct, one starts with the cloned genomic fragment (contained in an E. coli plasmid vector). Restriction digestion or other related methods can be used to cut out the internal sequences and replace them with the selectable marker gene. This is shown in Figure 1.4.

More recently, PCR-based methods have been used for the construction of the disruption fragment. In yeast, only about 40 bp of sequence are needed at each end of the disruption fragment in order for the crossover events to occur properly, but the sequence must be identical to the genomic target sequence. The PCR primers are used to amplify the selection gene and place a target site sequence at either end. Each primer consists of 40 bp of target site sequence at the 5' end followed by a short sequence homologous to the selection gene. The complete selection marker gene must be amplified, including the promoter and ORF. Longtine et al. (1998) describe plasmid constructs designed specifically to provide selection marker templates for PCR amplification. The kanMX6 resistance gene and the Saccharomyces pombe his5+ gene fused to appropriate S. cerevisiae promoter and terminator sequences as well as the S. cerevisiae TRP1 gene are available in this series. The kanMX6 and his5+ genes are particularly useful because they lack homology to S.

YGI1

Chromosome

Crossover

^ Crossover

I

HIS3

1

Transformation of his 3 host strain with disruption fragment Select for His+ transformants

H1S3

Figure 1.3 One-step gene disruption of YG11

Chromosome

Figure 1.3 One-step gene disruption of YG11

Cloned genomic fragment

HIS3 gene fragment

Disruption fragment

Figure 1.4 Construction of a disruption fragment using available restriction sites

Cloned genomic fragment

HIS3 gene fragment

Disruption fragment

Figure 1.4 Construction of a disruption fragment using available restriction sites cerevisiae genomic sequences and thus preclude the possibility of recombination at sites in the genome other than at the intended disruption site. When deleting a gene, it is best to remove sequences starting in the promoter and extending into the ORF or past the stop codon. This ensures that the gene has been functionally knocked out. If the transcription and translation start sites are not removed and the deletion is internal to the ORF, it is conceivable that some gene function could be retained.

Whether the traditional or the PCR-based method is used for one-step gene disruption, it is important to confirm that the event has occurred correctly. This can be done by Southern analysis or by PCR of genomic DNA using one primer that anneals to sequences outside the deleted region and one primer that anneals to internal sequences in the selection marker gene.

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