出芽酵母におけるタンパク質ベース継承の高速スクリーニング

The overall goal of this high-throughput yeast phenotyping is to screen for proteins that illicit a lasting phenotypic memory of passed over expression, as a proxy for protein-based inheritance. This method can help answer key questions in the yeast Prion field, such as, how many proteins can it illicit inheritance of this type. The main advantage of this technique is that you can screen many proteins and pathways in an unbiased fashion.

Start this procedure by growing the appropriate yeast cells in a 96 well plate at 30 degrees celsius, as described in the text protocol. Visually inspect the cultures to confirm that the cultures are saturated, cells should be visible by eye, at the bottom of each well. Use a liquid handling robot to prepare 384 well plates, containing 45 microliters of the appropriate medium per well.

First, dispense Scal-URA into the wells of a 384 well plate. Then dispense Scal-URA plus the stressor of interest into the wells of a second plate, 20 millimolar of manganese chloride is used as the stressor in this experiment. Thirdly, dispense SD-URA, which will not induce plasmid expression, plus manganese chloride into another plate.

Lower the 96 well plate containing the cultures onto the liquid handling robot and inoculate a one to four array of one microliter from each well of the 96 well plate, into four separate wells of each 384 well plate filled with a different media. Immediately place the plates with cells on a microplate stacker at room temperature and atmospheric carbon dioxide. Set the protocol for a 72 hour continuous loop, measuring the optical density at 600 nanometers with the microplate reader.

After the growth measurements, transfer one microliter per well of the SCal-URA induced cultures that experienced protein over-expression to new 384 well plates, containing 45 microliters per well of ST-URA medium that does not permit protein expression of the plasmid. In parallel, perform analogous inoculations of a second set of 384 well plate containing 45 microliters per well of ST-URA from the cultures that were grown in ST-URA in the presence of the stressor. Place the plates in a humidified chamber and grow the plates for 48 hours at 30 degree celsius to saturation.

After 48 hours, use the plates to re-inoculate one microliter per well in 384 well plates containing 45 microliters per well of ST-URA. Then, perform a separate re-inoculation of one microliter per well from the same source plate into a separate 384 well plate containing 45 microliters per well of ST-URA with the stressor. Immediately place the plates with cells of the microstacker at room temperature and atmospheric carbon dioxide and set the protocol for a 48 hour continuous loop, measuring the optical density at 600 nanometers with a microplate reader.

When the growth measurements are complete, use the plate reader software to export the time versus optical density at 600 nanometer measurements as an XY table. Group columns of the optical density measurements for each biological replicate together and calculate the mean. Create an XY plot of time versus optical density at 600 nanometers to generate growth curves.

For cultures that chose significant growth differences in response to a given stressor, dependent upon ancestral protein over-expression, take one microliter from each biological replicate, dilute in 10 milliliters of water and then plate 50 microliters on plates containing 5-FOA. Grow at 30 degree celsius for three days. If this results in too many or too few colonies per plate, adjust the dilution factor accordingly.

100 to 200 colonies per plate is ideal. Pick eight to 32 single colonies with autoclave toothpicks and pin them to 96 well plates containing 150 microliters of SDCSM per well. Place the plates in a humidified chamber and grow to saturation for 48 to 72 hours at 30 degrees celsius.

Use these cultures to inoculate two new sets of 96 well plates containing 150 microliters per well of SDCSM, both with and without the stressor. Place the plates on a microplate stacker and set the protocol to measure the optical density at 600 nanometers on a 48 hour continuous loop. After 48 hours, analyze the data as shown earlier and confirm that the significant growth differences seen earlier, are maintained after plasmid loss.

The cells that maintain the induced phenotypes are then tested for classic hallmarks of protein-based inheritance as described in the text protocol. This protein-based inheritance screen revealed the transient over-expression of the PSP1 open reading frame, drives a resistance to manganese chloride that was maintained for hundreds of generations in cells, long after over-expression had ceased. Prion predicting algorithms scored the end terminus of PSP1 as moderately Prion-like.

In contrast, the algorithms predicted virtually no significant Prion-like sequence features from most inducing proteins recovered on the screen, as shown by this representative analysis. Growth measurements of strains and SDCSM with manganese chloride, normalized to a corresponding naive PSP1 minus control, indicated that inhibition of the HSP104 disaggregase does not impair PSP1 dependent manganese chloride resistance whereas the removal of the HSP70 chaperone and the PSP1 gene, heritably eliminates this phenotype. All spores from single tetrads of crosses between strains, harboring the PSP1 dependent phenotypic state and naive strains, display manganese chloride resistance, indicating non-mendelian inheritance of the phenotype.

A comparison of the infectivity for protein transformation between mock lysates and PSP1 harboring lysates as the transmissible material, showed that over 53%of naive cells transformed with seeded PSP1, received the corresponding manganese chloride resistance phenotype. Once mastered, this technique can be done in a couple of weeks, including incubation steps and the preparation of most screening steps can be done in less than two hours per day, even with a large number of plates. While attempting this procedure, it is important to remember to plan exactly what you wanna test beforehand and keep in mind the scale of the experiment.

Now this is especially important to beginners of the technique. Following this procedure, other methods like those described in the text protocol can be performed to determine if the phenotypes observed in the screening display a true, Prion-like pattern of inheritance. After its development, this technique paved the way for researchers in the field of epigenetics to explore the breadth of Prion biology in sacralis service CI.After watching this video, you should have a good understanding of how to design a high throughput screen for new forms of protein-based inheritance in yeast, in both an unbiased and targeted fashion.

Don’t forget, that certain chemical stressors such as DNA damaging agents can be extremely hazardous and precautions, such as wearing personal protective equipment, should always be taken while performing this procedure.