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

PCR Methods Appl. 1994 Apr;3(5):272-7. doi: 10.1101/gr.3.5.272.

Abstract

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Activating Transcription Factor 1 / genetics
  • Activating Transcription Factor 1 / isolation & purification*
  • Activating Transcription Factors / genetics
  • Activating Transcription Factors / isolation & purification*
  • Base Sequence
  • Cloning, Molecular / methods*
  • DNA-Binding Proteins / isolation & purification*
  • Meiosis
  • Molecular Sequence Data
  • Phosphoproteins / genetics
  • Phosphoproteins / isolation & purification*
  • Polymerase Chain Reaction*
  • RNA / analysis*
  • Recombination, Genetic
  • Schizosaccharomyces / cytology
  • Schizosaccharomyces / genetics*
  • Schizosaccharomyces pombe Proteins / genetics
  • Schizosaccharomyces pombe Proteins / isolation & purification*

Substances

  • Activating Transcription Factor 1
  • Activating Transcription Factors
  • DNA-Binding Proteins
  • Pcr1 protein, S pombe
  • Phosphoproteins
  • Schizosaccharomyces pombe Proteins
  • atf1 protein, S pombe
  • RNA