The overall goal of this procedure is to determine the genetic control of translocation formation by single stranded kneeling in diploid budding yeast cells. This is accomplished by first inoculating cultures with single colonies of a specific genotype and growing them overnight. The second step of the procedure is to induce simultaneous double strand break formation by ho endonuclease on two chromosomes by the addition of galactose to the cultures.
Next dilution of these cultures are plated on selective and non-selective media. The final step is to determine translocation frequency by counting colonies arising on selective and non-selective media. Ultimately, results can be obtained that show genetic control of translocation formation by single strand, a kneeling through changes in the frequency of ho stimulated translocation formation in strains of different genotypes.
The implications of this technique extend toward the consequences of therapeutic radiation exposure of cancer patients because DNA double strand breaks and translocation formation are the primary consequences of this treatment Treatment. To begin the procedure for determining ho stimulated translocation frequencies inoculate 10 to 20 independent one milliliter cultures of Y peak glycerol lactate medium with single colonies of the desired genotype, incubate the cultures overnight at 30 degrees Celsius on a rotator. When the cultures have reached the desired cell density add 20%galactose to the cultures to a final concentration of 2%The galactose will induce expression of the ho endonuclease, which will direct double strand breaks at the history delta five prim allele on chromosome three and has three delta three prim allele on chromosome 15 incubate for four hours at 30 degrees Celsius on a rotator.
The next step is critical to the success of the procedure. Careful dilution and plating of the cultures is imperative. If you were to get translocation frequencies that are sufficiently reproducible to permit statistically significant comparisons between strains of different genotype After four hours plate appropriate dilution of the cultures onto medium lacking histidine as well as onto YPD td approximately 100 to 200 colonies per plate incubate plates at 30 degrees Celsius for two to three days.
When colonies arise, the translocation frequency can be determined. Plating efficiencies before and after induction of ho endonuclease expression will indicate if the presence or absence of either translocation chromosome affects the ability to survive DSB formation. That fragments one copy of both chromosome three and chromosome 15 to determine pre-induction plating efficiencies.
First, remove an aliquot of cells from each overnight culture and determine cell number by counting with a hemo cytometer. Next, use an appropriate dilution to plate approximately 100 to 200 cells onto YPD incubate for two to three days at 30 degrees Celsius until colonies arise. Add 20%galactose to the remaining cultures to a final concentration of 2%and incubate a 30 degrees Celsius for four hours to determine post induction plating efficiency.
Remove an aliquot of cells from each culture after four hours of induction and determine cell number by hemo cytometer Count plate approximately 100 to 200 cells using an appropriate dilution onto YPD incubate for two to three days at 30 degrees Celsius until colonies arise. Once colonies arise on the plates plating efficiency can be determined. Putative translocation bearing clones can be examined by southern blot.
This procedure uses genomic DNA prepared from a single hiss plus recombinant colony from each independent trial digest approximately four micrograms of each DNA sample with BAM H one restriction endonuclease separate BAM H one digested fragments on a 0.7%aros she and then transfer to a positively charged nylon membrane hybridized with a P 32 labeled probe obtained by random priming with a 1.8 KILOBASE BMH one BMH one genomic clone containing the HIS three gene. Visualize DNA fragments by auto radiography or phospho imaging. Another way to examine putative translocation bearing clones is by chromosome plot analysis.
Chromosomes are first prepared from selected hiss plus recombinants in aros plugs separate chromosomes on a 1%aros gel in a contour clamped homogeneous electric field or chef at 14 degrees Celsius. The following parameters are used first block 14 degrees celsius, 72nd switch time and 15 hours at six volts centimeter, second block, 14 degrees Celsius, 122nd switch time and 11 hours at six volts per centimeter. At the completion of the electrophoresis, visualize chromosomes by staining the gel with AUM bromide for 30 minutes, followed by irradiating with UV and detaining and distilled water transfer chromosomes to a positively charged membrane by capillary action.
Denaturing conditions hybridize with P 32 labeled probe obtained by random priming with a 1.8 kilobase bam H one bam H one genomic clone containing the hiss. Three gene visualize chromosomes by auto radiography or phospho imaging. The frequency of chromosomal translocations can be calculated by dividing the number of histamine phototrophic colonies by the total number of viable cells determined by plating onto YPD.
In this example, the calculated recombination frequency is about 3%The assay for ho stimulated translocation frequencies can be conducted using strains of different genotypes to compare differences in the ability to repair double strand breaks by single strand. An kneeling as shown in this representative graph. The translocation frequencies obtained both selectively and nons selectively with the RAD 51 delta null homozygote were statistically different from those obtained using the corresponding conditions with the wild type strain both pre and post induction plating.
Efficiencies are determined by dividing the total number of viable colony forming cells by the total number of cell bodies in the culture determined by hemo cytometer count as shown in this table pre and post induction plating. Efficiencies were not significantly different for wild type cells, which suggests that the presence or absence of either translocation chromosome does not affect the ability to survive. DSB formation.
Putative translocation bearing clones can be examined by genomic southern blot analysis. A graphical representation of relevant chromosomes before and after translocation formation is shown here. Sizes of restriction fragments containing relevant sequences generated by BAM H one digestion of genomic DNA from parent and recombinant strains and revealed on blots by hybridization with a 1.8 kilobase BAM H one.
His three genomic clone are listed in kilobase pairs for southern blot analysis. Genomic DNA is digested with BMH one endonuclease and hybridized to a P 32 labeled 1.8 kilobase. HIS three probe to visualize these diagnostic fragments.
0.8 kilobase HIS three delta 201.7 kilobase hiss three delta three prime four kilobase T 3 15 5 kilobase T 15 three and eight kilobase hiss three delta five prime fragments. Lane A is the parent diploid. Lane B is a hiss plus non-reciprocal translocation recombinant and lane C is a his plus reciprocal translocation.
Recombinant chromosomal BLO analysis can also be employed to examine translocation bearing clones. Intact chromosomes prepared in aros plugs are separated by chef electrophoresis and the gel stained with a iridium bromide and visualized under UV light. The separated chromosomes are then blotted to nylon and hybridized with the P 32 labeled 1.8 kilobase.
His three probe to visualize the 1.1 megabase intact chromosome 15.8 megabase T 15 three translocation chromosome 0.6 megabase T three 15 translocation chromosome and 0.3 megabase intact chromosome three. Lane A is the parent deployed. Lane B is A his plus non-reciprocal translocation recombinant, and Lane C is a his plus reciprocal translocation recombinant.
After watching this video, you should have a good understanding of how to determine the frequency of translocation formation following multiple ho stimulated double strand breaks.
