Mobility of yeast mitochondrial group II introns: engineering a new site specificity and retrohoming via full reverse splicing

Cell. 1997 Mar 21;88(6):865-74. doi: 10.1016/s0092-8674(00)81932-7.

Abstract

The mobile group II introns aI1 and aI2 of yeast mtDNA encode endonuclease activities that cleave intronless DNA target sites to initiate mobility by target DNA-primed reverse transcription. For aI2, sense-strand cleavage occurs mainly by a partial reverse splicing reaction, whereas for aI1, complete reverse splicing occurs, leading to insertion of the linear intron RNA into double-stranded DNA. Here, we show that aI1 homing and reverse splicing depend on the EBS1 (RNA)/IBS1(DNA) pairing and that target specificity can be changed by compensatory changes in the target site and the donor intron. Using well-marked strains to follow coconversion of flanking DNA, we show that homing occurs by both RT-dependent and -independent pathways. Remarkably, in most RT-dependent events, the reverse spliced intron is the initial template for first-strand cDNA synthesis.

Publication types

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

MeSH terms

  • Base Sequence
  • DNA, Complementary / analysis
  • DNA, Complementary / chemical synthesis
  • DNA, Fungal / analysis
  • DNA, Mitochondrial / physiology*
  • Endonucleases / genetics
  • Gene Expression Regulation, Enzymologic / genetics
  • Gene Expression Regulation, Fungal / genetics
  • Introns / genetics*
  • Mutagenesis, Site-Directed / genetics
  • Plasmids
  • RNA Splicing / genetics
  • Recombination, Genetic
  • Yeasts / enzymology
  • Yeasts / genetics*

Substances

  • DNA, Complementary
  • DNA, Fungal
  • DNA, Mitochondrial
  • Endonucleases