Silencing in Yeast
Efficient handling of multiple reactions is a crucial prerequisite for productive RNA differential display (DD) analysis. To identify transcriptional targets of the histone H3 Lys9-specific methyltransferase Clr4, we applied a multiformat modification of DD to compare between clr4 + and clr4 − transcriptomes of Schizosaccaromyces pombe. As a result, 14 differentially expressed bands were identified among 720 polymerase chain reaction (PCR) studied. The content of these bands was then analyzed by cloning, sequencing, and Northern analysis. In the final stage of verification, four Clr4 targets were isolated based on their expression in six Clr4 chromo and SET domain mutant strains. The step-by-step description of the multiformat DD provided below includes RNA purification, cDNA synthesis, 96-well PCR, electrophoretic separation of PCR products, isolation of DNA fragments from differentially expressed bands, and verification of candidate genes by Northern analysis.
Key WordsRNA differential display clr4 silencing yeast histone-specific methyltransferase transcriptional regulator
We thank Dr. W. Modi for advising on gel loading strategy. This work was supported in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract no. NO1-CO-56000. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does the mention of trade names, commercial products, or organizations imply endorsement by the US government.
- 1.Allshire, R. C. (1996) Transcriptional silencing in the fission yeast: a manifestation of higher order chromatin structure and function, in Epigenetic Mechanisms of Gene Expression (Russo V.E.A., Martienssen R.A., and Riggs A.D, eds.), Cold Spring Harbor Laboratory Press, Cold Spring Habor, NY, pp. 443–466.Google Scholar
- 2.Klar, A.J.S., Ivanova, A. V., Dalgaard, J. C., Bonaduce, M. J., and Grewal, S.I.S. (1998) Multiple epigenetic events regulate mating-type switching of fission yeast, in Epigenetics (Chadwick D.J. and Casdew G., eds.), Wiley and Sons, Chichester, UK, pp. 7–103.Google Scholar
- 8.Sambrook, J. and Russell, D. W. (2001) Molecular Cloning. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.Google Scholar