Lecture 24 - Genetic Manipulation

Lecture 24 - Genetic Manipulation - Lecture 24 Genetic...

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Lecture 24 - Genetic Manipulation - "Reverse" Genetics Forward Genetics: finding the genetic basis of a phenotype. Reverse Genetics: finding the phenotype(s) that derive from a genetic sequence. Baker's Yeast ( Saccharomyces cerevisiae ) "simple" unicellular eukaryote --has a nucleus and mitochondrion. Arguably, the most advanced experimental system with which to study basic eukaryotic cell biology and metabolism. Upsides: small genome, even for a unicellular fungus, (~12 Megabasepairs) encoding just ~6,000 genes. Note that the other fungus that we've covered Neurospora has ~35 Megabasepairs and ~10,000 genes Downsides: "missing" features characteristics of eukaryotes (e.g. no light perception, no circadian rhythm and no DNA methylation) Biochemical markers : As with Neurospora, isolating a mutant with a biochemical deficiency is feasible. Plate mutagenized wild type haploid yeast on synthetic complete (SC) medium (same as rich medium) replica plate onto synthetic complete minus amino acid (e. g. leucine). Identify colonies that grow on SC but fail to grow on SC -leucine. The mutant yeast strain is leu . As we previously covered, the loss-of-function in multiple genes in leucine biosynthesis will yield a leu phenotype. One commonly used auxotroph is leu2 --the " 2 " designating a particular leucine biosynthetic gene. Other popular auxotrophs are his3 (for histidine) and ura3 (for uracil). Genetic Manipulation : With yeast, we have two basic options for genetic transformation. 1) Under the right conditions, "naked" linear DNA is taken up by yeast cells and integrates into chromosomal sequence via homologous recombination. Genetic transformation is inefficient, which means that we need to select for rare (one in a million) transformants. The wild type gene of an auxotrophic marker is
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commonly used as a selection marker. To target integration of the selection marker to a particular chromosomal sequence, the selection marker DNA is flanked on both ends with DNA sequence that is homologous to the chromosomal insertion site. For successful integration, DNA homology in each flank must recombine with
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Lecture 24 - Genetic Manipulation - Lecture 24 Genetic...

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