[23] Transfer RNA import into plant mitochondria

Publisher Summary This chapter discusses the transfer (t) RNA import into plant mitochondria and the experimental procedures that have been used to identify nucleus-encoded tRNAs in plant mitochondrial (mt) tRNA populations and to study tRNA import into mitochondria in vivo . It describes the preparation of plant nucleic acids and enzyme extracts and the fractionation and identification of individual species in plant mt tRNA population. The genetic origin of individual plant mt tRNAs isolated by two dimensional polyacrylamide gel electrophoresis (2D-PAGE) can be determined using classical dot blots, which allow more probes to be studied simultaneously, or Southern blots, which also give some information on the gene copy number and on the size of the corresponding restriction fragments. These techniques are performed in the usual manner and according to the membrane manufacturer's instructions. Hybridization with 3'-end-labeled tRNAs is performed as described.