Silencing in yeast: identification of clr4 targets.
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.
Ivanov
Sergey V
SV
SAIC-Frederick, NCI Center for Cancer Research, Frederick, MD, USA.
Ivanova
Alla V
AV
D015152:Blotting, Northern; D018797:Cell Cycle Proteins / Q000235:genetics*; D018076:DNA, Complementary / Q000378:metabolism; D003851:Deoxyribonucleases / Q000378:metabolism; D004586:Electrophoresis; D004587:Electrophoresis, Agar Gel; D020869:Gene Expression Profiling / Q000379:methods*; D015966:Gene Expression Regulation, Fungal; D020868:Gene Silencing; D000076983:Histone Methyltransferases; D011495:Histone-Lysine N-Methyltransferase / Q000235:genetics; D008780:Methyltransferases / Q000235:genetics*; D009154:Mutation; D016133:Polymerase Chain Reaction; D011496:Protein Methyltransferases; D012313:RNA / Q000737:chemistry; D012568:Schizosaccharomyces / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins / Q000737:chemistry / Q000235:genetics*
D018797:Cell Cycle Proteins; D018076:DNA, Complementary; D029702:Schizosaccharomyces pombe Proteins; D012313:RNA; D000076983:Histone Methyltransferases; D008780:Methyltransferases; D011496:Protein Methyltransferases; D011495:Histone-Lysine N-Methyltransferase; C112744:clr4 protein, S pombe; D003851:Deoxyribonucleases