Selection of mutants

Mutants defective in particular enzymes can be selected if two conditions are fulfilled either the enzyme is not essential or an analogue of the normal substrate leads to lethal incorporation. 

Cells lacking thymidine kinase (TK- cells) can be isolated by treating cell cultures with high concentrations (30 jug/ml) of 5-bromodeoxyuridine, which kills cells containing the enzyme thymidine kinase due to incorporation of large amounts of the analogue into the cells DNA. 

5BUdr 5BdUMP - 5BdUDP - 5BdUTP - DNA thymidine kinase 

Mutants lacking, or with much reduced levels of the enzyme thymidine kinase survive as this enyzme is not essential. Thus the cell can make dTMP from dUMP using folic acid as the one carbon donor (Fig. 13.1). 

TK mutants arise at a very much higher frequency than would be expected for a diploid cell and the evidence suggests that while one of the alleles has been mutagenised, the other allele has been 

---

Selection of mutants was done by increasing the chloramphenicol concentration in liquid BG-11. Control and mutant strains were grown at a light intensity of 30 pmol photons-m"2 s at 45°C with air/C02 bubbling. 

Oberly, T.J., Bewsey, B.J. and Probst, G.S. (1987). A procedure for the CHO/HGPRT mutation assay involving treatment of cells in suspension culture and selection of mutants in soft agar. Mutation Res. 182 99-111. 

The methods of selection of mutant TK and HGPRT-defective cells from the permanent cellular lines of domestic animals are developed (SPEV, TR, and PO-2). 

Low-level resistance to chloramphenicol may emerge from large populations of chloramphenicol-susceptible cells by selection of mutants that are less permeable to the drug. Clinically significant resistance is due to production of chloramphenicol acetyltransferase, a plasmid-encoded enzyme that inactivates the drug. 

Table 8.1 Positive and negative cross-resistance of PS II herbicides in a selection of mutants of various organisms...

Selection of mutants in mammalian cells in culture involves several technical variables, including isolation protocol, phenotypic expression, time before selection, cell density that permits maximal mutant recovery, drug concentration, and culture media. 

OUABAIN A chemical of botanic origin that inhibits sodium-potassium-activated ATPase in cell membranes, thereby being toxic to the cell the selection of mutants that are resistant to the toxic effects of ouabain provides the basis of a mutation-detection system in mammalian cells. 

Major advantages that bacteria hold for biochemists are the ready availability of mutants coupled with a short generation time. Compared with whole animals, animal cells in culture have a relatively short generation time and so a major effort has gone into the production and selection of mutants of animal cells and their use in the study of somatic cell genetics. 

The added attraction of the transition from myoblast to myotube is the synchrony with which differentiation occurs in vitro. Myogen-esis will occur in primary cultures of skeletal muscle (e.g. 6.12) but can also be induced in diploid myoblast lines (Richter and Yaffe, 1970) which has allowed the selection of mutants (Chapter 13) that exhibit drug resistance or temperature-sensitive differentiation (Loomis et al., 1973 Somers et al., 1975). Holtzer et al. (1975) and Fiszman and Fuchs (1975) have developed a myoblast line transformed with a temperature-sensitive virus. At the permissive temper-... 

Almost all the phototrophic bacteria strains used in hydrogen production are from the genera Rhodobacter (and the most used species is R. sphaeroides). Some species formerly designated as Rhodopseudomonas have been classified as Rhodobacter in recent classification criteria (information from Jun Miyake). Maybe some strains designated as Rhodopseudomonas and Rhodospirillum should be re-classified under new criteria, but in this column is presented the name suggested by the authors. Adaptation of strains to particular media or the selection of mutants, maybe is not enough to define new species. 

O Donnell, J., L. Gerace, F. Leister and W. Soffer. (1975) "Chemical selection of mutants that affect alcohol dehydrogenase in Drosophila. II Use of 1-pentyn-3-ol." Genetics 79 73-83. 

J. M. Widholm,in F. Constabel and I. K. Vasil (Eds), Cell culture and somatic cell genetics of plants 4. Selection of mutants which accumulate desired secondary compounds. Academic Press, San Diego, 1987, pp. 125-137. 

In higher plants altered photosynthetic electron transport in the triazine-resistant mutants (2, 3) has been correlated with slower growth and lower yield (jT). Experiments with Chlamydomonas, a unicellular alga, suggest that trlazine resistance is not necessarily associated with an alteration of the photosystem II electron transport kinetics ( 5). Selection of mutants resistant to other classes of photosystem II inhibiting herbicides, (e.g. dluron, bromacll) should also be feasible based on successful Isolation of such mutants in Chlamydomonas (16). 

---