Unusual mtDNA Control Region Length Heteroplasmy in the COS-7 Cell Line.

The COS-7 cell line is a workhorse of virology research. To expand this cell line's utility and to enable studies on mitochondrial DNA (mtDNA) transcription and replication, we determined the complete nucleotide sequence of its mitochondrial genome by Sanger sequencing. In contrast to other available mtDNA sequences from Chlorocebus aethiops, the mtDNA of the COS-7 cell line was found to contain a variable number of perfect copies of a 108 bp unit tandemly repeated in the control region. We established that COS-7 cells are heteroplasmic with at least two variants being present: with four and five repeat units. The analysis of the mitochondrial genome sequences from other primates revealed that tandem repeats are absent from examined mtDNA control regions of humans and great apes, but appear in lower primates, where they are present in a homoplasmic state. To our knowledge, this is the first report of mtDNA length heteroplasmy in primates.

The mitochondrial genome sequence of the BS-C-1 cell line is at odds with the reported derivation from Cercopithecus aethiops.

The BS-C-1 cell line widely used in virological research was reportedly derived from the African green monkey Cercopithecus (Chlorocebus) aethiops. We used Sanger sequencing to determine the full nucleotide sequence of mtDNA in BS-C-1 cells. The mitochondrial genome in this cell line is 16,456 bp long and has an organization typical of that in other mammalian genomes with 37 genes, including 2 rRNAs, 22 tRNAs, and 13 polypeptide-encoding genes. Surprisingly, this genome is most closely related to that of Chlorocebus pygerythrus, which does not agree with the reported derivation of this cell line from C. aethiops. Another unexpected observation is the presence of mtDNA length heteroplasmy within the MT-ND6 gene, with the variant containing a stretch of 8 Cs encoding a functional gene, whereas the variant containing 9 Cs encodes a frameshifted gene. It is unclear whether the latter variant is nonfunctional or whether it is corrected by programmed translational ribosomal -1 frameshifting. The availability of the full mtDNA sequence for the BS-C-1 cell line should increase its utility by enabling studies on mtDNA transcription and replication.

Methods for Efficient Elimination of Mitochondrial DNA from Cultured Cells.

Here, we document that persistent mitochondria DNA (mtDNA) damage due to mitochondrial overexpression of the Y147A mutant uracil-N-glycosylase as well as mitochondrial overexpression of bacterial Exonuclease III or Herpes Simplex Virus protein UL12.5M185 can induce a complete loss of mtDNA (ρ0 phenotype) without compromising the viability of cells cultured in media supplemented with uridine and pyruvate. Furthermore, we use these observations to develop rapid, sequence-independent methods for the elimination of mtDNA, and demonstrate utility of these methods for generating ρ0 cells of human, mouse and rat origin. We also demonstrate that ρ0 cells generated by each of these three methods can serve as recipients of mtDNA in fusions with enucleated cells.