The steady-state levels and metabolic properties of mitochondrial tRNAs have been analyzed in Hela cells and correlated with the function of the tRNAs for organelle-specific protein synthesis. DNA excess hybridization experiments utilizing separated strands of mitochondrial DNA (mtDNA) and purified tRNA samples from exponential cells long-term labeled with [³²P] orthophosphate have revealed a steady-state level of 6 x 10⁵ tRNA molecules per cell, with three-fourths being encoded in the heavy (H)-strand and one-fourth in the light (L)-strand. Hybridization of the tRNAs with a panel of M13 clones of human mtDNA containing, in most cases, single tRNA genes and a quantitation of two-dimensional electrophoretic fractionations of the tRNAs have shown that the steady-state levels of tRNAF and tRNAV are two to three times higher than the average level of the other H-strand-encoded tRNAs and three to four times higher than the average level of the L-strand-encoded tRNAs. Similar experiments carried out with tRNAs from cells labeled with very short pulses of [5-³H] uridine have indicated that the rates of formation of the individual tRNA species are proportional to their steady state amounts. Therefore, the 15-fold to 60-fold higher rate of transcription of the tRNAV and tRNAF genes (transcribed with the rDNA transcription unit) relative to the other H-strand tRNA genes (transcribed with the whole H-strand transcription unit) and the 13-fold to 20-fold higher rate of transcription of the L-strand tRNA genes relative to the H-strand tRNA genes (other than tRNAV and tRNAF genes) are not reflected in the rates of formation of the corresponding tRNAs. The available data indicate that the majority of tRNAV and tRNAF transcribed from the rDNA transcription unit are degraded as they are excised from the primary transcripts. It also seems likely that the majority of the L-strand-encoded tRNAs are degraded before they are excised from the short-lived polycistronic transcripts. Furthermore, a role of the aminoacyl tRNA synthetases in stabilizing the different tRNA species at relatively uniform levels is suggested. A comparison of the steady-state levels of the individual tRNAs with the corresponding codon usage for protein synthesis, as determined from the DNA sequence and the rates of synthesis of the various polypeptides, has not revealed any significant correlation between the two parameters.
In other experiments, isolated human mitochondria containing a mitochondrial DNA (mtDNA)-coded chloramphenicol resistance marker were injected at an average dose of less than one into sensitive human cells partially depleted of their mtDNA by ethidium bromide treatment. Under selective conditions, the mitochondria became established in the recipient cells with a frequency greater than 2 to 3 x 10⁻³). A rapid and, in some cases, complete replacement of the resident mtDNA by the exogenous mtDNA took place in the transformants, as shown by multiple mtDNA and nuclear DNA polymorphisms. Intracellular mtDNA selection played a crucial role in this replacement, with significant implications for mitochondrial genetics.
