A chloramphenicol-resistant mutant, isolated from mouse A9 cells, was enucleated and fused with a nucleated chloramphenicol-sensitive mouse cell line. Resultant fusion products, cytoplasmic hybrids (or "cybrids"), were selected as resistant to chloramphenicol, and had the nuclear markers and chromosome complement of the chloramphenicol-sensitive parent. These cybrids appeared at the high frequency of 2-8 per 104 cells plated. Neither parent produced any colonies when plated under identical selective conditions. Fusion between enucleated chloramphenicol-sensitive cell fragments and the chloramphenicol-sensitive cell produced no resistant colonies, suggesting that chloramphenicol resistance is not due to an increase in the ratio of cytoplasm to nucleus. Furthermore, fusions between resistant and sensitive nucleated cells produced resistant hybrids at a frequency 100 times less than that of resistant cybrids. Thus, these stable chloramphenicol-resistant cybrids result from the fusion of a chloramphenicol-resistant cytoplasm with a chloramphenicol-sensitive cell. It is proposed, therefore, that chloramphenicol resistance is a cytoplasmically inherited characteristic in this mouse cell line.It has been established that mitochondria contain, in addition to their own DNA, the biochemical apparatus for translation and transcription of this genetic information. Studies on the genetics of mitochondria are most advanced in yeast, where several antibiotic resistance and respiratory deficiency mutations are known to be coded in mitochondrial DNA (mt-DNA) and cytoplasmic genetic recombination has been demonstrated (1, 2). Mammalian cell mtDNA is smaller in size than yeast mtDNA. Hybridization studies have shown that mammalian mtDNA codes for ribosomal RNA and 12 distinct 4S RNAs (3).A genetic approach to mtDNA function in mammalian cells requires the isolation of mutants with altered mitochondrial properties, and the demonstration of the cytoplasmic inheritance of such properties. Chloramphenicol (CAP) inhibits mitochondrial protein synthesis in human HeLa cells (4), and this laboratory has recently described a HeLa mutant whose mitochondrial protein synthesis is resistant to CAP (5, 6). Further, CAP resistance in yeast is coded by mtDNA (7). However, no method has been described as yet to demonstrate cytoplasmic inheritance in mammalian cells.A CAP-resistant mutant of the mouse line A9 has been isolated in this laboratory in a manner similar to that for the HeLa CAP-resistant mutant. This paper describes the experimental evidence that CAP resistance in mouse cells is cytoplasmically inherited. MATERIALS AND METHODSStrains and Culture Conditions. Strains A9 and LMTKare subclones of mouse L-cells, a line of aneuploid fibroblasts. Strain A9 is deficient in hypoxanthine phosphoribosyltransferase (HPRT; EC 2.4.2.8) activity and resistant to 8-azaguanine; LMTK-is deficient in thymidine kinase (TK; EC 2.7.1.75) activity and resistant to 5-bromodeoxyuridine (BrdU) (8,9). Both lines are sensitive to 50 gg/ml of CAP and do not...

The cytoplasmic inheritance of human chloramphenicol (CAP) resistance has been demonstrated by removing the nuclei of cells of the CAP-resistant HeLa strain 296-1 (enucleation) and fusing them to a CAP-sensitive HeLa strain lacking nuclear thymidine kinase. Plating the fusion products in bromodeoxyuridine and CAP resulted in the growth of about 150 colonies/106 parent cells plated. Permanent cell lines (cybrids) grown from such fusions have been designated HEB.

This study indicates that t-PA and PAI-2 may play a significant rôle in the periodontal tissue destruction and tissue remodeling and that t-PA and PAI-2 in GCF may be used as clinical markers to evaluate the periodontal diseases and assess treatment.

No abstract

Abstract. New mutant yeasts resistant to the antibiotics chloramphenicol and mikamycin were isolated. They are mitochondrial mutants, characterized by several criteria as cytoplasmically determined. Biochemical studies show that amino acid incorporation into protein in vitro by mitochondria isolated from cells resistant or sensitive to mnikamycin or chloramphenicol is inhibited by these antibiotics. Although aerobically-grown resistant strains of Saccharomyces cerevisiae are not affected by mikamycin or chloramphenicol, it is found that the mitochondrial protein-synthesizing system of anaerobically grown cells is inhibited in vivo. Cross resistance among the antibiotics chloramphenicol, mikamycin, erythromycin, lincomycin, carbomycin, and spiramycin is reported. All erythromycin resistant mutants, unlike the others, are resistant to erythromycin in vivo and in vitro. The results indicate that some of the cytoplasmic mutations (mikamycin and chloramphenicol resistance) are expressed at the mitochondrial membrane, whereas others (erythromycin resistance) possibly reflect changes in mitochondrial ribosomal proteins. We further suggest that conformational changes, either in the membranes or ribosomes, are likely to account for the observed antibiotic cross resistances.The mitochondrial protein synthesizing system of Saccharomyces cerevisiae is specifically inhibited, in vivo and in vitro, by a number of antibacterial antibiotics: these include chloramphenicol, lincomycin, erythromycin, carbomycin, and spiramycin. '2 Yeast grown in the presence of these antibiotics lack the particulate mitochondrial cytochromes a, a3, b, and cl, thus are unable to grow on nonfermentable substrates. An erythromycin-resistant strain of S. cerevisiae showing cytoplasmic inheritance has been isolated and partially characterized in this laboratory.3 4 In vivo and in vitro, its mitochondrial protein synthesizing system is unaffected by high levels of erythromycin. We have suggested, by analogy with bacterial mutants, that a change in the response of the mitochondrial protein-synthesizing system to erythromycin is a result of an alteration in a mitochondrial ribosomal protein. 3'4 In this communication we report the isolation and some biochemical properties of two new cytoplasmically-determined mutant types of S. cerevisiae resistant to mikamycin and chloramphenicol. In contrast to the cytoplasmically-deter-1233