Laboratory Notebook 7: RNA 70-116, 1988 - 1989

The notebook begins with Greider treating RNA strands with a MNase enzyme, which split the strand in half. Greider then ran the RNA strands through a gel, thereby creating six ladders of DNA each with corresponding bands and graphed the sensitivity of MNase onto the RNA strand. Then Greider tested the MNase enzyme on a sodium chloride compound and DNA. Greider found that there was something in the DNA and RNA strands that seemed to inhibit the MNase enzyme.

After conducting the MNase experiment, Greider tried to see if there were differences in the pH effected telomere activity of the strands. Previous experiments by other researchers showed that a pH of 8.5 seemed to be optimum. The pH ratio, she believed, would minimize the effects of the fluctuating pH when reacting with telomeres. After examining the effects of pH fluctuations, Greider used different enzymes each with a pH ranging from 6.0-8.5. Greider then froze the remaining enzymes for later tests, in order to understand pH related stability and then loaded a gel.

The next set of notes revolved around a group meeting. Much of the discussions were about reconstitution, as well as the 159 RNA in yeast, which led to a successful expression of RNA. The discussion was productive as Grieder learned two different approaches to reconstitution. The first is the standard approach, which separates RNA from protein. The second utilized the MNase enzyme to destroy the RNA. However, with this method there still will be some RNA bits left. After doing experiments on both methods, Greider obtained odd results from the second approach. It seemed that MNase didn’t fully break down the RNA strand.

Greider also did assay’s and gels that separate the RNA from protein, mated different Tetrahymena vegetated cells, and removed RNA by spinning the cells. She finished the procedd by starving them. Grieder then loaded the RNA into a gel with 121 volts. The resulting gels were too variable, so Grieder attempted a dilution and loaded a dye to mark the bands of RNA and graphed the results.

In another experiment, Greider separated RNA strands from a protein mixture and utilized the MNase enzyme. Greider then changed the variables of the experiment by adding Urea gel to the RNA in different ways in order to separate it from the protein mixture. In her next group meeting, they discussed repeated use of RNase on oligonucleotides in RNA sequence. Also Greider told the group that she obtained a viable oligomer nucleotide that could encode the added G4T2 repeats found in telomere. Grieder had ran the repeat through a gel and found a problem. The experiment then created 4000-6000 clones and two libraries worth of information were negative for the gene.

Greider summarized her findings for the group. Greider wanted to show that the enzyme that she had found had an important role in telomere production as she had previously postulated. However, so far the direct evidence has been shown to exist only in ciliates like Tetrahymena. Greider also wanted to know the structure and function of RNA in relation to telomere work. Also Greider began probing yeast species to see if the enzyme that she had found worked in there telomere production.