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  1. Genetika molekuler (6) Sutarno

  2. 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. • Integrating v. low efficiency stable 1 copy/cell • CEN high efficiency stable low copy (1-2 / cell) • 2m high efficiency stable high 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 • it’s 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 • It’s 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 wouldn’t 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: insertionvsreplacement of part of the ORF) • Advantage: less likely to be functional • Can’t 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 • Don’t 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