RNA Extractions:

In order to further characterize the dominant-negative mutants I had collected, I decided to screen for defects in the synthesis of rRNAs by using RNA extraction to find the relative levels of rRNA in the cells. I extracted the RNA from the four mutants that did not grow at all on galactose, the three mutants that grew slowly on galactose, the mutant that was contaminated with mold, the control containing the original pBM258T/YTM1 plasmid, and two controls containing a gap repaired pBM258T/YTM1 with an unmutagenized insert (one from each of transformations 2 and 3).

1. For the first RNA extraction, I forgot to shift the cells from glucose media to galactose. Each sample was resuspended in 20ul dH2O. This extraction served as a practice. I ran my samples on a 1% agarose gel (not very RNase free) to get an idea of how much of each sample to load on a gel in the future. 20ul of each sample seemed to be more than enough; 10ul would have also worked. Because my gel was not RNase free, the bands came out as very big smears—the RNA had been degraded.

2. For the second RNA extraction, I grew up my cells in 5ml of C-ura overnight and then shifted them to C-ura+gal for another overnight. For controls, I grew up two cultures of JWY3400 (the parent strain) in YEPD and shifted one to YEPGal. I inoculated the other culture into YEPD. I harvested the cells during log phase growth (OD = ~0.4), extracted the RNA, and resuspended my final pellets in 20ul dH2O. I ran 20ul of each sample on a 1% agarose gel. However, as I was loading the gel, my samples started leaking out of the wells. I ran the gel and found that most of the sample had left the wells. The only RNA was located in the wells still; nothing had run down the gel.

I also took the OD260 of each sample to find out how much RNA was present in each sample. I diluted 2ul of each sample into 500ul dH2O for a 1:250 dilution. The levels of RNA present in each sample varied greatly. Table 1 shows the concentrations of RNA in each sample.

3. When I repeated the RNA extraction the third time, I made all new solutions in order to ensure that everything was RNase free. I grew up the cells in 5ml C-ura overnight and shifted has I had during the second RNA extraction. I used the same controls as well. I extracted the RNA and resuspended in 20ul. This time I ran only 10ul of each sample on a 1% agarose gel. When I loaded the gels, I had no problems with dye leaking out of the wells as I had seen before. However, after I started running the gel, I noticed that some of the dye had started leaking out. I let it run for 15 minutes and then checked to be sure that some of the RNA sample had traveled into the gel. Once I saw RNA in the gel, I let it run for 2 more hours at 80V.

The RNA gel shows that most of the RNA in the wells had floated out, as shown in figure 2. The RNA that I had seen in the gel did not move in the two hours that the gel was running.

Again, I took the OD260 of a 1:250 dilution of each of the samples. As before, I could see no clear correlation between the level of RNA in the sample and the growth rate of the cells on galactose. The levels of RNA varied greatly throughout the samples. Table 1 shows the RNA concentrations of the samples.

Figure 2: 1% Agarose Gel of RNA Extractions From Putative ytm1 Mutants