Requirement for Cell Dispersion Prior to Selection of Induced Azaguanine-Resistant Colonies of Chinese Hamster Cells
AUTOR(ES)
Myhr, B. C.
RESUMO
With V79 Chinese hamster cell cultures treated with a mutagen, the maximum frequency of colonies resistant to 8-azaguanine (AZG) was attained when the cells were dispersed after a suitable expression time before adding the selection medium. V79–4 cells were exposed to 500 µM MMS, 7 µM AFAA, or 10 µM MNNG and allowed to multiply before being reseeded at 4 x 104 cells/60 mm dish and selected with 10 µg/ml AZG. Maximum frequencies of 4 x 10-5, 4 x 10-4, and 2.4 x 10-3 were obtained about 100, 130, and 200 hrs after exposure to MMS, AFAA, and MNNG, respectively. The maximum frequencies following MMS or MNNG treatments were about 10-fold greater than those obtained when induction and selection of AZG-resistant colonies were performed in the same culture dish. The reseeding of treated cells eliminated the possibility of metabolic cooperation within mosaic colonies of wild-type and mutant cells and achieved expression of the induced changes before intercolony crossfeeding reduced the frequency of resistant colonies.—AZG-resistant colonies were selected in medium containing dialyzed fetal bovine serum, and the selection medium was replaced at least twice. Both serum dialysis and selection medium replacement were necessary for consistent achievement of background frequencies of resistant colonies near 10-6. Reconstruction experiments with AZG-resistant V79 lines showed that the efficiency of recovery of resistant cells in the selection medium was constant over a range of 0–20 colonies observed/dish. A mixed population of V79 and AZG-resistant cells was also correctly analyzed by the procedure used in mutagenesis studies.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=1213312Documentos Relacionados
- Mammalian Cell Genetics I. Selection and Characterization of Mutations Auxotrophic for L-Glutamine or Resistant to 8-Azaguanine in Chinese Hamster Cells IN VITRO
- Defective Guanine Uptake in an 8-Azaguanine-resistant Mutant of Salmonella typhimurium
- Genetics of Somatic Mammalian Cells. IV. Properties of Chinese Hamster Cell Mutants with Respect to the Requirement for Proline
- Cytoplasmic requirement for peroxisome biogenesis in Chinese hamster ovary cells.
- Expression of Purine Overproduction in a Series of 8-Azaguanine-Resistant Diploid Human Lymphoblast Lines