How can the vectors be integrated into yeast genome as the rate of homologous recombination is low?
Actually homologous recombination is not that low in yeast. You can facilitate plasmid integration by creating a double strand break in the plasmid within a region of homology to chromosomal DNA (selectable marker if the plasmid is complementing an auxotroph for example). The cut greatly enhances integration frequency, and directs the plasmid to the homology region defined by the cut. Classic work from Terry Orr-Weaver in the Szostak lab defined these parameters decades ago.
Hi there,
Yeast integrative plasmid is devoided of any replication origin which means the isolated circular plasmid will be unstable in the cellular context because unable to replicate and therefore will be lost after a few cell division. Now if this plasmid is encoding for a protein essential for the cell survival (selection marker) then cell will integrate the plasmid sequence into its genome (this is the simplest event to allow the stabilization of the plasmid content) and therefore selection pressure will encourage and select the homologous recombination event between homologous régions of the plasmid and genome.
Actually homologous recombination is not that low in yeast. You can facilitate plasmid integration by creating a double strand break in the plasmid within a region of homology to chromosomal DNA (selectable marker if the plasmid is complementing an auxotroph for example). The cut greatly enhances integration frequency, and directs the plasmid to the homology region defined by the cut. Classic work from Terry Orr-Weaver in the Szostak lab defined these parameters decades ago.
You can do it by homologous recombination. You need to have a region of homology cloned in your vector, and you can transform it with single double strand break in the homology region. It will get integrated into the yeast genome by HR. And selectable marker will be used to for screening the integrated clones.
In this case the promoters count like homologous sequences for the plasmid? Malcolm Whiteway
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