Laboratory Notebook 8: In Vitro 97-123/RNA 122-135, 1989

The notebook begins with diagrams of RNA structure. The structure noted due to abundance of Urasil nucleotide. Greider also constructed diagrams of elongation of C4A2, its translocation with G4T2 and then subsequent elongation. Greider reviewed her previous RNA and invitro experimentsand fractionalization studies. Grieder also remarked on each experiment, and made notes on which ones involved gels and titrations.

In group meeting, Greider was still working on reconstitution study from earlier. Found that she had little success separating RNA from protein having tried different avenues such as a northern blotting technique on the RNA strand. The notebook also contained correspondences between Dave, a colleague of Greider's, and Greider. The letters detailed methods to extract cells from yeast culture as well as Yeast RNA gene, which has been referenced earlier. Dave thought the Yeast RNA can be transcribed, and to help Grieder he detailed the transcription reaction.

Greider told Dave that the single strand RNA gene was efficiently transcribed and had even enclosed a figure of transcription. Beyond the correspondence Greider conducted several gel studies, dilution and titration studies, and graphed the results of each. She also conducted southern and northern blot tests, and had run into problems because Grieder only had little RNA.

An interesting note within this notebook is the RNase H Knockout Experiment the Greider conducted. RNase H has a unique property of digesting the RNA from any DNA/RNA duplex. She preinclubated the RNase H and Tested against telomere activity. It must have been to much RNAse H because Grieder redid the experiment with less telomerase.

In another experiment, Greider mixed T2G4 with different levels of pBR. pBR is known for excising certain areas of a 5 prime- 3 prime sequence. Grieder graphed the levels of pBR and also created an even more specific graph of the 3 prime levels. Also graphed different oligo titrations, highlighting certain points of the graphs. Afterwards she made new buffers and loaded gels for assay and graphed the columns with corresponding numbers of base pairs.

In another group meeting, Greider decided to collaborate with a colleague. They were looking at telomere lengths in fibroblast cultures. They cultured cells for increased number of passages and looked at the length of the telomere. They saw that the telomere length get shorter making the signal weaker. The repeat sequence, T2AG3, gets lost at senescence. They also never saw activity of reconstitution. In the end they found that they needed to test several thing. First is to analyze if certain enzymes limiting telomeric length. the second is how long ar oligonucleotides and to possibly utilize better substrates. The last is to create a schematic representation of their proposed repeat organization, using human telomeres as the model. In order to undertsnd the over all effect of the experiment they graphed the results.

Decided to conduct a probe experiment on MNase enzyme. It was tested in both hot and cold conditions. They dried down the hot side and electroblotted the cold side. Then loaded a gel with the results from both. They repeated the experiment but with more MNase, probed with (C4A2)2. They ran titration/dilution with crude (T2G4)4 Oligo. Also ran gels of RNA extracted from titration and dilution experiment, and on reconstructed RNA.