Record Details
In vitro transcription in the yeast: Saccharomyces cerevisiae
ScholarsArchive at Oregon State University
| Field | Value |
|---|---|
| Title | In vitro transcription in the yeast: Saccharomyces cerevisiae |
| Names |
Ide, Gregory James
(creator) Van Holde, K. E. (advisor) |
| Date Issued | 1980-10-02 (iso8601) |
| Note | Graduation date: 1981 |
| Abstract | The structure and transcriptional activity of intranuclear and isolated chromatin from logarithmically growing yeast cells has been compared to chromatin from cells which have entered the stationary phase and ceased growing. Both chromatins show a similar nucleosomal repeat pattern and a 160 by repeat size when digested with staphlococcal nuclease. The rate of DNase I digestion of growing phase is greater than in stationary. Growing phase nuclei are also 5 to 20 times as active as stationary in the amount of endogenous transcription. Analysis of elongating transcripts indicates the transcriptional differences between growing and stationary are due to differences in in vivo initiation. The DNase I susceptability and transcriptional differences noted in nuclei are maintained in sucrose gradient isolated oligonucleosomes and mononucleosomes from the two states. As an adjunct to structural and transcriptional studies of yeast,a rapid technique for isolation of yeast nuclei has been developed. Briefly, the method consists of layering of the 18% ficoll lysate prepared by the method described in Lohr and Ide (1979), on an isopycnic density gradient of 1M sorbitol, 0.5mM CaC1₂ dissolved in a solvent of 35% Percoll (Pharmacia) 65% H₂O, pH 6.5. The gradient is pre-formed before loading by spinning 34ml of the gradient solution contained in a 50ml tube in an SS-34 angle rotor at 37,000 xg for 50 minutes. Six ml of the 18% ficoll lystate is diluted with 6m1 1M Sorbitol 0.5mM CaC1₂ and then layered on this gradient. Nuclei are banded free of cell debris by a 7,500 rpm spin in an HB4 swinging bucket rotor for 15 minutes. The resulting band of nuclei is washed by dilution with 2 volumes 1M Sorbitol, 0.5mM CaC1₂ pH 6.5 and pelleted at 4300 xg for 5 minutes. Nuclei isolated by this method will incorporate 20 to 40 picomoles UTP into RNA per ug template DNA in a 15 minute synthesis. The nuclei are substantially free of cytoplasmic contamination as measured by alcohol dehydrogenase activities. Transcription initiation in isolated yeast nuclei by endogenous RNA polymerase has been studied using nucleoside 5'-[γ-S] triphosphates as affinity probes. In vitro initiated RNA can be separated from bulk RNA on a mercury agarose affinity column. Activity that transfers the [γ-S] group to other nucleotides or other RNA molecules (often troublesome in other systems) cannot be detected. Analysis of the in vitro initiated RNA shows that 5S and pre t-RNA are initiated in vitro by endogenous RNA polymerase III. Endogenous RNA polymerase III also initiates a discrete distribution of RNA species as large as 28S. The RNA populations initiated with 5'-[γ-S] adenosine 5' triphosphate and 5'[γ-S] guanosine 5' triphosphate are different. |
| Genre | Thesis/Dissertation |
| Topic | Chromatin |
| Identifier | http://hdl.handle.net/1957/42116 |
