Genetika Molekuler (5) Sutarno

Lecture #4 Notes (Yeast Genetics) LECTURE 4: CLONING AND MANIPULATING GENES IN YEAST Basically, we use the same techniques that were used in bacteria. First we need to understand the types of vectors available in yeast. Integratingv. low efficiencystable1 copy/cell CENhigh efficiencystablelow copy (1-2 / cell) 2mhigh efficiencystablehigh copy (~50 copies / cell) Selection for transformants that have taken up the DNA requires a dominant marker (in bacteria this is usually drug resistance markers) In yeast the selection is typically for complementation of a nutritional defect CLONING GENES WHEN YOU HAVE A RECESSIVE MUTATION Transform the mutant strain (Ts- Ura-) with a yeast WT genomic DNA plasmid library (CEN library typically is used first) Select for Ura+ transformants Screen for those transformants that reverse (complement) the mutant phenotype its relatively easy….takes only ~2,700 colonies (insert size of 25 kb) for 99% assurance of covering the whole genome (only 5 plates might be enough!)(or they might not) Why you might not get the gene Not in the library No restriction sites nearby Too many internal restriction sites Near CEN, TEL, repeats etc (hard to clone regions) Lethal in bacteria Library could be made from a mutant yeast strain The phenotype is lousy Leaky Reverts frequently Need a good screen positive vs negative growth phenotypes… examples: drug resistant mutant transform, screen for sensitivity Ts- mutant transform, screen for growth at non- permissive temperature Its a big gene (less likely to be full length clones due to size restriction and more sites) How to get around those problems? Use a different library Try to complement another phenotype More transformants Map and clone by phone You might have cloned the gene, but maybe not! Revertants Dosage suppressors Duplicated genes or with overlapping functions (example: histone loci) (all are interesting, but still have to determine which is occurring) TESTING IF YOU CLONED THE CORRECT GENE Clues: Does it complement all the phenotypes? Are there multiple isolates of the same genomic region? Is the reverted phenotype plasmid-dependent? Lose the plasmid and ask if the mutant phenotype returns either by non-selective growth or on 5-FOA plates URA3 = a decarboxylase that converts 5-FOA to 5 Flourouracil (toxic) A VERY useful reagent that used very frequently, since there is a strong, clean, positive selection for both URA3+ and ura3- Ura3+(5-FOAs)select on SC-Uracil plate Ura3-(5-FOAr)select on 5-FOA Isolate the plasmid from the reverted colony, and re- transform the purified plasmid (not the library) back in to the original strain Integrate Does it direct integration to the correct chromosomal locus? Integration in yeast: Yeast has very high levels of recombination Linear ends are more recombinogenic than internal DNA sequences Integration occurs at the homologous chromosomal locus The procedure Integrate into a wild type strain Result is duplicated WT gene, with URA3 between Cross with the mutant (ura3-) strain Dissect tetrads If it is the correct gene, URA3+ will now be integrated (tightly linked) at the YFG locus Therefore, all tetrads will have 2 Yfg+ Ura+ spores and 2 Yfg- Ura- spores (PDs) Q: If it integrates somewhere else (presumably unlinked), what would be expected? A: expect 1:1:4 (lots of recombinant Ura+ Yfg- and Ura- Yfg+ spores) OK, the gene is on the plasmid. What next? Sequence the ends of the inserts Find the ORFs in SGD (any obvious candidates?) Subclone obvious candidate ORFs (or all of them if necessary). How do you clone the gene when your strain has a dominant mutation? Since the mutation is dominant, it wouldnt help if you transformed in the wild type gene from a genomic library. You need to first make a genomic library from the dominant mutant strain. Example: you have a dominant mutation that makes the strain red. Make a genomic library from the red strain Transform it into a wild type (white) strain Select transformants Look for red colonies. Still have to confirm that it is the correct gene by (1) re- transformation and (2) integration. Then subclone to determine which ORF. MANIPULATING THE CLONED DNA Creating a true null WHY?: Critical for clean interpretation. It tells you what a complete loss of function phenotype is. Assumptions are made when interpreting standard recessive or dominant mutations. The only really cleanly interpretable allele is a true null. The history of creating clean nulls: Disruption using a gene fragment (1982) Advantage: The disruption is marked by a selectable marker Problems:still can be functional Parts are duplicated, so excision is possible One step disruption (two versions: insertion vs replacement of part of the ORF) Advantage:less likely to be functional Cant generate WT by recombination Problem:somewhat limited by the available restriction sites PCR-based precise nulls Advantages:simple…just PCR and transform Fast…a clean null in about a week Precise replacement of any desired segment Dont even need the gene cloned What if the gene is essential? Do it in a diploid. What is expected in tetrads if the gene is essential? 2 viable spores, 2 dead, and the viable ones should be auxotrophic for the null marker Important: need to rescue viability with the WT gene on a plasmid. Now you can work somewhat with the null strain. THE PLASMID SHUFFLE Nulls are important, but they are not always the most useful allele. (especially if they are dead) EXTREMELY POWERFUL TECHNIQUE !!!! Two main uses: To test whether a homolog from another species will complement knockout of an essential gene (use the Zoller figure) To isolate new alleles (the REAL power of the technique) Create a mutagenized library Hydroxylamine PCR Mutator bacteria Oligos (a la Cormack and Struhl) Advantage: allows isolation of extremely rare mutations due to the targeted mutagenesis (impossible to mutagenize the whole cell, targeting only a single locus) VERY important for reverse genetics (allows isolation of Ts mutants if the gene is essential) Allows creativity in finding more interesting alleles for TBP, polymerase-specific mutants mutants that recognize a non-TATA sequence (altered specificity) mutants defective in activating specific genes mutants that are more active than WT TBP If your gene has multiple functions, can select for mutants defective in only one of those functions. (e.g. are the functions genetically separable?) Let the yeast tell you what is important !!! Practically a limitless technique…the limits are your cleverness in finding a phenotype / selection that will generate the most informative mutants. Two-step gene transplacement Used to introduce new mutations from a plasmid into the correct genomic location 1) first linearize within the gene, and integrate, resulting in URA3 between the two copies 2) next select for 5-FOAr colonies that have looped out one of the copies 3) select/screen for the phenotype of interest Combined with plasmid shuffle, we can select for a phenotype to identify new mutants, and then put it back into its normal genomic location. Gap rescue of mutant alleles A method for rescuing a mutant locus from a genomic location onto a plasmid. Start with a CEN plasmid in which the area of interest is removed (by a simple restriction digest), leaving some homology with the adjacent genomic regions on the plasmid. Transform into the mutant strain The gap gets filled in with the homologous sequence from the chromosomal locus, with the yeast doing all the work. Sequence the mutant allele. Using these techniques we can now do almost anything that we want to, and can go through either a complete reverse or classical genetics strategy: Classical: Isolate mutants using different combinations of selections, targeting them to identify specific classes of genes if appropriate Classify: dominant/recessive, complementation groups Map the mutation Clone the gene Interpret its broad role in the process being studied Reverse: Knock out any gene (or part of a gene) precisely and rapidly Select for rare, interesting, and informative mutations with the plasmid shuffle Replace the WT gene with any mutants (selected or specifically created) by transplacement Rescue genomic mutations onto plasmids Overexpress any gene under regulated or constitutive promoter