RNA associated with a heterodimeric protein that activates a meiotic homologous recombination hot spot: RL/RT/PCR strategy for cloning any unknown RNA or DNA.
The ade6-M26 mutation in the fission yeast Schizosaccharomyces pombe creates a meiotic homologous recombination hot spot. We have achieved 40,000-fold purification of a heterodimeric DNA-binding protein, Mts1/Mts2, that activates the recombination hot spot. Physical studies suggested the presence of a third subunit. It is demonstrated here that RNA molecules of approximately 210 nucleotides copurified with the heterodimer. To characterize the RNA component, it was necessary to develop a new strategy for cloning of the unknown, low-abundance, partially degraded RNAs that were present in purified Mts1/Mts2 protein preparations. The strategy uses RNA ligase to add DNA oligonucleotide priming sites to the RNA for subsequent reverse transcription and PCR (RNA ligase, reverse transcription-PCR, or RL/RT/PCR). This cloning procedure could be applied to the cloning of any unknown RNA or DNA molecules. Because the cDNA clones obtained from Mts1/Mts2 were largely heterogeneous, it seems likely that the RNAs copurified as a result of tight but nonspecific interactions with the heterodimeric protein.
Wahls
W P
WP
Fred Hutchinson Cancer Research Center, Seattle, Washington 98104.
D051697:Activating Transcription Factor 1 / Q000235:genetics / Q000302:isolation & purification*; D051696:Activating Transcription Factors / Q000235:genetics / Q000302:isolation & purification*; D001483:Base Sequence; D003001:Cloning, Molecular / Q000379:methods*; D004268:DNA-Binding Proteins / Q000302:isolation & purification*; D008540:Meiosis; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction; D012313:RNA / Q000032:analysis*; D011995:Recombination, Genetic; D012568:Schizosaccharomyces / Q000166:cytology / Q000235:genetics*; D029702:Schizosaccharomyces pombe Proteins / Q000235:genetics / Q000302:isolation & purification*
D051697:Activating Transcription Factor 1; D051696:Activating Transcription Factors; D004268:DNA-Binding Proteins; C097701:Pcr1 protein, S pombe; D010750:Phosphoproteins; D029702:Schizosaccharomyces pombe Proteins; C106740:atf1 protein, S pombe; D012313:RNA