Drug-resistant colonies

Assay of Resistant Colonies for Levels of HPRT Activity 

Large-sized AG- or TG-resistant colonies randomly isolated at the end of the selection period were dispersed with trypsin, suspended in culture medium, and seeded into a 30-ml flask and into two 60-mm dishes, one of which contained a sterile coverslip. To avoid the possibility of culturing a minor fraction of HPRT cells that might be transferred along with the AG- or TG-resistant colony (cf. Jacobs and DeMars 0 cells in the flask were grown to confluence in selective medium. Crude cell extracts were prepared according to the method of Milman et The level of HPRT enzyme activity, i.e., 

Determination of Maintenance of Resistant Phenotype in the Absence of Selection 

A portion of the cells dispersed from these same resistant colonies was plated into nonselective culture medium and allowed to grow for approximately 10 generations. Cells were then trypsinizcd and plated at cloning densities into three different types of media (1) HAT medium, i.e., Ham s FIO that had been prepared without HX and thymidine but was now supplemented with 30 mM HX (H), 0.1 aminopterin (A), and 30 nM thymidine (T) and 10% FCS (2) nonselective culture medium, i.e., FIO lacking HX but supplemented with 10% FCS and (3) the AG or TG selective medium described in Section 2.1. Growth in HAT medium requires a cell to possess HPRT activity (or, alternatively, to be resistant to aminopterin). Growth in AG or TG selective medium requires a cell to lack HPRT activity or to have altered uptake of these purines. After 3 weeks, the dishes were stained and the number of clones in each set determined. 

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Number of drug resistant colonies formed following culture of cells obtained from collagenase-dissociated organs with metastatic foci composed of tumor cells transfected with a selective vector. 

The first requirement is that the drug-resistant colonies be mutants, which has never been proven directly (via sequencing) for any mammalian mutation assay. However, indirect evidence that is consistent with a mutagenic mechanism can be obtained. Drug-resistant colonies should maintain their resistance indefinitely in the absence of the selective agent. Drug resistance should correlate strictly with a specific biochemical defect. 

The isolation of the gene coding for H. volcanii DHFR (Rosenshine et al, 1987) deserves special mention. As mentioned above, H. volcanii is very sensitive to the antifolate drug trimethoprim. Spontaneous resistant colonies arise at frequencies of 10 10-10 9. The molecular basis for the resistance in all the resistant mutants studied so far is... 

Growth of L. donovani in completely defined medium has facilitated the isolation of clonal variants resistant to cytotoxic purine analogues In these experiments 10 promastigotes were plated under selective conditions, either 5uM formycin b or luM tubercidin, and from one to about fifty resistant colonies appeared per plate. The isolation of such clonal drug-resistant variants from large populations of parasites is a necessary prelude to the genetic dissection of the purine salvage pathways unique to these parasites. 

Quinidine (6) and quinine (7) are diastereomeric quinoline alkaloids obtained from Cinchona spp. Quinidine (6) is included in many pharmacopeias for its antiarrhythmic effects.Quinine was the first antimalarial drug and served as an effective remedy for this deadly infectious disease in colonial times, making European settlement in many tropical and subtropical parts of the world possible.Owing to the development of resistance to synthetic antimalarials, quinine is still reverted to some extent for this... 

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