The overall goal of this procedure is to generate specific mutations in histone genes at their endogenous chromosomal locations in budding yeast cells. This method can be used to determine the contribution of specific histone residues in a variety of processes that use chromatin as the substrate, such as DNA transcription, every combination. The main advantage of this technique is that specific histone genes can be targeted for mutagenesis despite the fact that in yeast, each histone protein is encoded by two highly homologous, non-allelic genes.
After generating a yeast strain with the target histone gene knocked out and replaced by URA3, generate PCR products for two partially overlapping fragments of the target histone gene by setting up the following reaction for the first half of the gene. For the second half of the gene, set up a similar PCR reaction using different primers. Place the reactions in a thermocycler and set up the following program.
Run 20 to 50 microliters of the PCR reactions on a 0.9%low melting point agarose gel in TBE buffer. Then use a clean razor blade to cut the PCR products out of the gel and transfer each section to a 1.5 milliliter tube. Store the agarose pieces at 20 degrees Celsius until ready to use.
To prepare template DNA for the PCR reactions to generate full size PCR products, melt the agarose sections in a heat block at 65 degrees Celsius for five minutes. Vortexing the tubes every one to two minutes to help with melting. Transfer a set amount of melted agarose from each sample into a single Microcentrifuge tube and mix the tube by vortexing.
Use this as the template for fusion PCR and store at 20 degrees Celsius until use. To amplify a large quantity of full sized PCR product, set up six PCR reactions each with the following reagents. Place the tubes in the thermocycler and run the reactions with the following settings.
Concentrate the PCR products through precipitation and use them for cotransformation with the backbone plasmid according to the text protocol. To screen for the loss of the URA3 gene as a result of PCR product integration, replica plate cells from plates 1 to 20 by removing the plate lid and pressing the plate containing colonies onto sterile velvet. Transfer the cells from the velvet to a 5-FOA plate by pressing the plate on the velvet.
Incubate the plates at 30 degrees Celsius for two days. Then carefully inspect the plates for growth. After streaking and incubating candidate colonies on YPD plates according to the text protocol, replica plate each YPD purification plate to a fresh YPD plate, to a dropout plate lacking uracil to check for the loss of the URA3 gene, and a second dropout plate to monitor for the presence or absence of the backbone plasmid.
Following incubation, identify a colony from each candidate sample that is growing on the YPD plate but not growing on either dropout plate. Restreak these colonies onto fresh YPD plates. As say for proper integration of the mutant allele using PCR according to the text protocol.
This figure shows the PCR results for generating two partially overlapping HHT2 fragments with the desired mutations that ultimately result in an AGA to GAA codon change producing an R53E mutation. The result from a fusion PCR reaction that generated the full sized PCR products is illustrated here. The full sized PCR products contained 195 and 220 base pair regions homologous to the regions upstream and downstream of the HHT2 open reading frame respectively to drive homologous recombination.
In this example transformation, the full sized PCR products were cotransformed with the HIS-3 marked plasmid and transformants were selected on plates lacking histidine. HIS positive colonies were then screened for 5-FOA resistance. Examples of candidates as well as samples unlikely to represent the desired integration events are shown.
12 candidate samples were identified and subjected to PCR analysis for replacement of the URA3 gene with mutant PCR products. Four candidates showed integration of a PCR product at the correct location. One of these candidates in shown in lane four.
Since the AG2GA mutation generates a new EcoR1 restriction site, digestion with EcoR1 was carried out on one of the successful integration PCR samples to demonstrate that the mutant sequence had indeed been incorporated into the genome. Once all reagents are available, it should be possible to obtain yeast cells harboring the desired histone mutations in the time frame or 15 to 20 days. Once the desired mutation has been generated in the host strain, cells expressing the target histone protein solely from the mutagenized gene can be obtained through appropriate genetic crosses.
While attempting this procedure, it's important to remember that the perimeters for the PCR reactions may need to be adjusted based on the nature of the specific primers used in the experiments and the expected sizes of the PCR products. Although this procedure is well suited for histone gene mutagenesis as it allows for targeting a specific histone gene despite the presence of a second highly homologous, non allelic gene, it can also be used to generate mutations at other genomic locations. After watching this video, you should have a good understanding of how to generate targeted NC2 histone mutations in budding yeast using a combination of PCR approaches and yeast genetics techniques.
Don't forget that exposure to UV light can be extremely hazardous, and protective eyewear, shields, and clothing should always be used while working with a UV transilluminator. Also wear gloves, goggles, and protective clothing throughout most steps of the procedure.
