Sequence analysis of mitochondrial chloramphenicol resistance mutations in Chinese hamster cells

Abstract

A series of mitochondrially inherited chloramphenicol-resistant (CAP-R) mutants were isolated in Chinese hamster cells. To determine whether the Chinese hamster CAP-R mutations were homologous to those isolated in mouse and human cell culture systems, we determined the nucleotide sequence of the region of the mitochondrial 16S rRNA gene spanning the peptidyl transferase-encoding region for eight CAP-R mutant lines in addition to the parental wild-type line. Three main conclusions are drawn from these studies. (1) Although the region of the gene encoding the peptidyl transferase domain is highly conserved relative to that of mice and rats, the contiguous sequences show less conservation. This sequence divergence not only includes the accumulation of single base pair replacements, but also the presence of small insertions or deletions. (2) For six of the CAP-R mutants, heteroplasmic single base pair changes were detected. These mapped to the same sites within the peptidyl transferase domain as the mutations found previously in mouse and human CAP-R mutants. (3) Two Chinese hamster CAP-R mutants, both with an unusual drug resistance phenotype, did not carry any mutations within the CAP-R peptidyl transferase domain. However, both carried a heteroplasmic mutation at the position corresponding to nucleotide 2505 of the mouse 16S rRNA gene, a site predicted to map within a stem/loop structure attached to this key domain of the ribosome. This is the first evidence for mitochondrial CAP-R mutations that map outside the peptidyl transferase region.

Nucleotide changes in mitochondrial 16S rRNA gene from different mammalian cell lines.

“…3). This mutation was heteroplasmic as the mtDNA populations contained both wild-type and mutant copies of the rRNA gene, as observed in other chloramphenicol-resistant mammalian cells (Howell and Lee, 1989;Howell and Kubacka, 1993) and this was verified by restriction fragment analysis after the digestion of PCR-amplified region II DNA with HinfI whose recognition sequence corresponds to the mutation site (data not shown). The results demonstrate that the mtDNA of this line has undergone a single nucleotide change in the rRNA gene.…”

“…This deletion was detected not only in V79 cells maintained in our laboratory but also in another V79 cell line obtained newly from other laboratory and in CHO-K1 cells originated from ovary cells. These results, therefore, indicates that there is an error in the V79-cell sequence registered in GenBank (Howell and Kubacka, 1993). At the nucleotide position 118 of the region I, C to T transition was also observed in CHO-K1 cells (Fig.…”

“…One of the readily obtainable genetic marker for mtDNA mutation is the resistance to chloramphenicol which inhibits mitochondrial protein synthesis selectively by binding to the peptidyl transferase domain of 16S rRNA in mammalian cells (Branlant et al, 1981). It has been reported that the phenotype of chloramphenicol resistance is mitochondrially inherited and determined by a single base substitution in 16S rRNA gene in mammalian somatic cells in vitro (Kearsey and Craig, 1981;Blanc et al, 1981;Howell and Kubacka, 1993).…”

“…Total DNA was isolated by standard phenol/chloroform extractions (Sambrook et al, 1989) from about 1 × 10 7 cells per cell line which have been cultured in α -modified Eagle minimal essential medium supplemented with 10% fetal calf serum and antibiotics at 37°C in a humidified atmosphere of 95% air/5% CO 2 as described previously (Ikushima, 1990). The target region within mitochondrial 16S rRNA gene was amplified by PCR using two sets of primers reported by Howell and Kubacka (1993) (Fig. 1).…”

Novel point mutations in mitochondrial 16S rRNA gene of Chinese hamster cells.

“…(Blanc et al, 1981a and1981 b;Kearsey and Craig, 1981;Koike et al, 1983;Slott et al, 1983;Howell and Lee, 1989;Howell and Kubacka, 1993;Hashiguchi and Ikushima, 1998). To gain insight into the spontaneous mutagenesis of mammalian mtDNA, we have analyzed the spontaneous alteration of nucleotide sequences in the mitochondrial 16S rRNA genes from the CAP-R mutants isolated in Chinese hamster V79 cells without any specified mutational treatment.…”