(44) is microconidiating, fluffy, inositolless, isoleucineless, and valineless.

Transcription

1 THE EFFECT OF PHOTOREACTIVATION ON MUTATION FREQUENCY IN NEUROSPORA' JEANETTE SNYDER BROWN Stanford University, Stanford, California Received for publication April 1, 1951 Kelner (1949a) first reported that mutations to phage resistance induced in Escherichia coli by ultraviolet light could be reversed by immediate irradiation with white light. In addition he noted that end-point mutations in contrast to zero-point mutations were not reversed by visible light (1949b). At the same time Novick and Szilard (1949) reported that light-reactivation of E. coli reduced the number of phage resistant mutants found after treatment with ultraviolet light. Recently Newcombe (195) has tested for streptomycin resistant mutants of E. coli after photoreactivation. His results are essentially similar to the studies on phage resistance. These results indicated that the killing of the bacteria and the induction of mutations might be due to the same chemical effect produced by the ultraviolet irradiation. In view of the importance of this finding to our understanding of the mechanism of action of ultraviolet radiation, it was desired to test the effect of photoreactivation on another entirely different mutation. The mutation selected was the reversion of the inositol locus in Neurospora crassa. Giles and Lederberg (1948) studied the adaption from inositol dependence to independence using macroconidia of a Neurospora, strain no. 3741, and found that reverse mutation occurred at the inositol locus, and that the frequency of this mutation could be increased by ultraviolet or X-radiation. This reverse mutation was followed in our experiments by plating treated uninucleate microconidia. EXPERIMENTAL METHODS Strains from three different parental backgrounds were used, each containing the same inositolless allele. Strain no. 522-CL-3-5A is microconidiating, inositolless, and colonial, and can therefore be plated without sorbose. Unfortunately, it has a high spontaneous reversion frequency to inositol independence. Strain no (44) is microconidiating, fluffy, inositolless, isoleucineless, and valineless. This isolate has a low spontaneous reversion frequency at the inositol locus and is fairly satisfactory. The minimal medium was supplemented with isoleucine and valine when testing for inositol reversion. A related strain isolated from a cross of strains 8743a and 3741A by R. C. Fuller in our laboratory proved to be even more suitable. This strain, no. -12, is microconidiating, fluffy, and inositolless. Fries minimal medium (Ryan et al., 1943) was used in all experiments. Either I Part of a thesis submitted in partial fulfillment of the requirements for the Ph.D. This work was done under the supervision of Professors A. C. Giese and E. L. Tatum at Stanford University. 163 Downloaded from on October 16, 218 by guest

2 164 JEANETrE SNYDER BROWN [vol. 62 one per cent sucrose or glycerol, and vitamins were added as the experiment demanded. Neither wildtype nor mutant grew well in appropriately supplemented glycerol liquid minimal although they grew quite well on these media solidified with 1.5 per cent agar. Sucrose, however, supported excellent growth in suitable liquid minimal. At first one-tenth per cent sorbose (Tatum et al., 1949) was added to the plating media when colonial growth was required. Later it was found that better germination could be obtained with.5 per cent sorbose when glycerol was used. Preparation of the material for irradiation was as follows: An abundant supply of mycelium was grown in sucrose complete liquid medium in a shake flask at 3 C for two days. A small amount of the mycelium was spread on sterile filter paper in a Buchner funnel. This paper was immediately transferred to a petri dish containing a one-quarter inch layer of glycerol complete, agar medium. Microconidia were produced plentifully in two to three days at C. When cultures were kept at a high humidity during conidiation, most of the conidia formed about the same time. Suspensions containing 5 X 18 microconidia per ml could be obtained from two such petri dishes by scraping from the plate, filtering through cotton to remove the mycelia, and washing by centrifugation with 2 ml of water. The method of irradiation used with strains no. 522 and no was as follows: A 5 cm diameter petri dish containing 1 to 2 ml of a conidial suspension in water was placed on a magnetic stirrer under a WL3 Westinghouse sterilamp for the desired time of exposure to ultraviolet light. After ultraviolet irradiation, an aliquot of the suspension was removed for the determination of the viable count by plating. The stirred suspension was exposed for 3 minutes to white light from a 1 watt General Electric mercury spot light at 5 cm distance from the dish. A crystallizing dish containing water to a depth of 6 inches was inserted between the light and the suspension to remove a large part of the infrared radiation. Maximlum survival was obtained with this dose of white light. In later experiments with strain no., one-tenth ml of a conidial suspension was placed in the center of a petri dish containing.5 per cent sorbose, solid medium either with or without 1,g per ml inositol. The suspension was evenly spread over the agar, except for a narrow band shaded by the side of the dish with a glass rod. Such a plate was placed under the sterilamp for the required dosage. It was then either incubated immediately in the dark at 3 C or first illuminated for one hour at 8 inches under a fluorescent light fixture containing two 1 watt "daylight" bulbs. Duplicate plates were always prepared for each dilution of the suspension and for each irradiation dosage. The advantages of the latter method were that each plate was an irradiation experiment, and less time elapsed between ultraviolet irradiation and dark incubation or white light irradiation. The organisms appear to be more sensitive to ultraviolet injury when treated by this method than when supended in water, but this is probably due to the lower mean intensity caused by absorption of the ultraviolet wavelengths by the medium. Downloaded from on October 16, 218 by guest

3 1951] MUTATION FREQUENCY IN NEUROSPORA 165 RESULTS AND DISCUSSION Table 1 presents the data on survival before and after light reactivation on minimal and supplemented media. In each experiment with strain no., about 5 of the colonies growing on minimal were picked to liquid minimal to test their inositol independence further. About one-third continued to grow in serial transfer. The failure of two-thirds of these colonies to grow was probably TABLE 1 The survival and number of reversions in microconidia of three Neurospora ino8itolle8s isolates following different dosage8 of ultraviolet radiation before and after light reactivation ISOLATE NUMEBR 522-C3-5A DOSAGE ULTRA- VIOLZT ERGS/NM With inositol 6,4, 41, 3, 66, 1,145, 1, 78,, <1, <1, 42 <1 184,, 8, 157, 26, 144,5, 9,5, 915, 58, 1,75 2,5 DARK SURVIVORS Without inositol < <1 < <1 Reversion frequency in 16 viable cells > With inositol 2,1, 1,85, 1,377, LIGHT SURlVIVORS 285, 26,8, 13,, 1,35, 1,98 117,5, 5,, 26,, 12,7, 91,, 52,,,, 1,65, 2,5, WWithoutl Iinositol <1 due to experimental difficulties. It was not due to a contamination of the original minimal medium with inositol or to a release of inositol into the medium by the organisms killed by the ultraviolet irradiation since as many colonies appeared at the 1/1 as at the 1/1 dilution. In itself this latter phenomenon would bear investigation. The apparent mutation frequency will vary, depending upon the dilution considered, but the values are consistent for a given strain and dilution in repeated experiments. Ryan and Lederberg (1946) encountered a similar situation during a study of reverse-mutation and adaption in a leucine Reversion frequency in 16 viable cells Downloaded from on October 16, 218 by guest

4 166 JEANETTE SNYDER BROWN [VOL. 62 less Neurospora. They concluded that heterocarbons with both reverted and leucineless nuclei were formed, and that the leucineless nucleus inhibited the reverted nucleus in the presence of a small amount of leucine. However, Giles and Lederberg (1948) studied a parent of the same inositolless Neurospora we were using, no. 3741, and found that inositol did not appear to cause selection against inositol independent nuclei. From the data in table 1, it must be concluded that the mutation frequency in light reactivated organisms is considerably less than in the dark survivors of ultraviolet irradiation, but it does not fall to the spontaneous mutation frequency. Most but not all of the ultraviolet induced mutations would be absent from material which had been irradiated immediately with visible light. The data given in table 2 show the effect of varying dosages of white fluorescent light following 1,5 ergs per mm2 of ultraviolet irradiation. Strain no. TABLE 2 The survival and number of reversions in Neurospora inositolless microconidia following uutraviolet radiation and increasing exposure to white light DOSAGE ULTRAVIOLET VITI H COUNT REVEON FREQUENCY ERGS/NM6TS 1 M LIGET With inositol Without inositol IN 16 VIABLE CELLS minuts o 56,5, , ,5, ,15, ,3, ,, and the agar plate method were used again. It has been suggested that mutated cells may not be reactivated as readily as cells which have sustained only cytoplasmic damage. Thus the mutation frequency would appear to be less after photoreactivation. The experiment presented in table 2 was conducted to test this hypothesis. If the mutants are not being reactivated, their absolute number should remain the same no matter how large is the dosage of white light. Actually, the absolute number of mutations increased with increasing amounts of white light. The increase is not of the same order as the increase in viability, and therefore an over-all decrease in mutation frequency results. In the light of this evidence, it seems likely that the mutation is prevented or reversed by white light exposure immediately following ultraviolet to a significant degree. SUMMARY Reversion at the inositol locus of Neurospora crassa was used to measure mutation frequency with and without photoreactivation. It was found that the high Downloaded from on October 16, 218 by guest

5 1951] MUTATION FREQUENCY IN NEUROSPORA 167 mutation frequency induced by ultraviolet light was lowered, but not to the original spontaneous frequency, by immediate exposure to visible light. The evidence thus indicates that mutation and lethality are due in part to the same effect of ultraviolet irradiation. REFERENCES GILES, N. H., JR., AND LEDERBERG, E Induced reversions of biochemical mutants in Neurospora crassa. Am. J. Botany, 35, KELNER, ALBERT 1949a Experiments on light-induced recovery of bacteria from ultraviolet irradiation injury. Abstracts of papers presented at the 49th General Meeting of the Society of American Bacteriologists, p. 14. KELNER, ALBERT 1949b Photoreactivation of ultraviolet-irradiated Escherichia coli, with special reference to the dose-reduction principle and to ultraviolet induced mutations. J. Bact., 58, NEWCOMBE, H. B. 195 Photoreversal of the mutagenic effect of ultraviolet light in E. coli. Genetics, 35, 682. NovIcK, A., AND SZILARD, L Experiments on light-reactivation of ultraviolet inactivated bacteria. Proc. Natl. Acad. Sci., U. S., 33, RYAN, F. J., BEADLE, G. W., AND TATUM, E. L The tube method of measuring the growth rate of Neuro8pora. Am. J. Botany, 3, RYAN, F. J., AND LEDERBERG, J Reverse-mutation and adaption in a leucineless Neuro8pora. Proc. Natl. Acad. Sci., U. S., 32, TATUM, E. L., BARRATT, R. W., AND CUTTER, V. M., JR Chemical induction of colonial paramorphs in Neurospora and Syncephalastrum. Science, 19, Downloaded from on October 16, 218 by guest