Silencing in Yeast

Identification of Clr4 Targets
  • Sergey V. Ivanov
  • Alla V. Ivanova
Part of the Methods in Molecular Biology book series (MIMB, volume 317)


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 Words

RNA 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.


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Copyright information

© Humana Press Inc. 2006

Authors and Affiliations

  • Sergey V. Ivanov
    • 1
  • Alla V. Ivanova
    • 1
  1. 1.SAIC-FrederickNCI Center for Cancer ResearchFrederick

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