Components of the Genetic Repair Mechanism are not confined to the Nucleus

Abstract

IT has been shown that radiation-sensitive mutants in separate genes, which presumably affect different steps in the repair of damaged DNA, can complement in Escherichia coli¹, or yeast² and Ustilago diploids. Could such strains complement each other's deficiencies in heterokaryotic cell ? Such complementation would not be expected if the repair mechanism was completely confined to the nucleus. Recently, mutants of Neurospora two or three times more sensitive to the lethal effect of ultraviolet light than wild type have been reported to complement in heterokaryons³. We have tested this further with three radiation sensitive (uvs) mutants of Ustilago maydis which are 10–30 times more sensitive to ultraviolet light than wild type cells, and which are believed to be deficient in ability to repair damaged DNA⁴. These mutants are recessive, unlinked and complement in diploid cells. Heterokaryotic cells were obtained using the conditions devised by Puhalla⁵ which allow strains of opposite mating type to fuse to form hyphal cells containing nuclei of opposite mating type, although we used a different medium (Ustilago liquid complete medium supplemented with 1.7 per cent Difco cornmeal agar). Groups of heterokaryotic cells were removed with a needle from compatible matings between strains with like or different uvs mutations, and spread on a plate of complete medium. The plate was irradiated with 960 ergs/mm² ultraviolet light from a standard germicidal lamp. This dose kills about 20 per cent of wild type cells and at least 98 per cent of uvs cells. After overnight incubation, 150 heterokaryotic cells of each type were scored for viability under a low power microscope. On this medium viable heterokaryons grow to form a microcolony consisting largely of haploid cells. The parent strains each carry complementing auxotrophic markers, and so it is easy to show that the surviving colonies are not diploids by replica plating them to unsupplemented medium on which only diploids will grow. The results of the tests are shown in Table 1.