Mutation Research, 28 (1975) 183-189 ((~ Elsevier Scientific Publishing Company, A m s t e r d a m - - P r i n t e d in The Netherlands

183

P O S T - I R R A D I A T I O N M O D I F I C A T I O N OF R A D I A T I O N - I N D U C E D H E T E R O A L L E L I C R E V E R S I O N IN D I P L O I D YEAST: E F F E C T . O F NUTRIENT BROTH

B. S. RAO, M. S. S. MURTHY, N. M. S. REDDY, P. SUBRAHMANYAM AND U. MADHVANATH

Division of Radiological Protection, Bhabha Atomic Research Centre, Trombay, Bombay-4oo o85 (India.) (Received July 9th, 1974) (Revision received December iSth, I974)

SUMMARY

The effect of post-irradiation growth in complete rich medium on the expression of the reversion to arginine-independence induced by g a m m a and alpha radiation in a heteroallelic diploid yeast strain (Saccharomyces cerevisiae BZ34) has been studied. During the post-irradiation treatment the reversion frequency increased, reached a peak at about 9 ° min and decreased thereafter reaching a constant value for treatment periods exceeding 6 h. As determined b y the increase in number of budding cells, extensive DNA synthesis took place in cells incubated only in the nutrient medium and not in the omission medium. Hence the observed increase in the reversion frequency is explained on the basis that post-irradiation DNA synthesis is necessary for the expression of gene conversion. The decrease in the reversion frequency for continued treatment with yeast extract, peptone, dextrose (YEPD) is related to the fact that only one daughter of the post-irradiation first cell division is a revertant. The broth effect was not lost when the irradiated cells were first incubated for 9 ° min in arginine-less medium and then transferred to the broth. Similarly, the broth effect persisted even at doses high enough to induce considerable division delay. These results suggest that the radiation-induced pre-conversional lesions are not susceptible to repair by alternative pathways.

INTRODUCTION

Post-irradiation treatment is one of the techniques widely used in bacteria and yeast for studying the intracellular mechanisms underlying the repair of radiationinduced lesions in DNA. Several workers have shown t h a t the UV-induced pre-mutational lesions are subiect to modification by post-irradiation treatmentS, 2°. In bacteriaS,8, 9, yeast 1~ and Neurospora 17, the UV-induced m u t a n t yield depends upon the ability of post-irradiation incubation medium to support protein synthesis. These Abbreviation: YEPD, yeast extract, peptone, dextrose.

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authors concluded that the pre-mutational lesions can be fixed by initiating rapid protein synthesis immediately after irradiation, which prevents the repair of pre-mu tational lesions. This conclusion is supported by the observation that the post-irradiation incubation of bacteria in a medium which cannot support rapid protein synthesis results in a decline of the mutation frequency-~-~, "°. WITKIN AND T H E I L ~1 have shown that not all types of mutation are susceptible to post-irradiation modification but probably only 'point' mutations are stabilized and fixed by immediate post-irradiation protein synthesis. KADA e¢ al. ~ have shown that some of the X-ray-induced mutations are refractory to post-irradiation treatment. While the earlier studies were on point-type mutations, gene conversion in diploid yeast studied in this investigation, is a genetic modification brought about by a recombinational process which by itself is also a repair process of the DNA damage produced by radiation ~. Irurther, gene conversion is a non-reciprocal event which is expressed only in one daughter cell after the first post-irradiation cell division. In this paper the effect of post-irradiation treatment in nutrient broth on the frequency of the radiation-induced reversion to arginine independence has been studied, with a view to understanding the mechanism of induction and expression of this type of genetic change. MATERIALS AND METHODS

Strain

The diploid strain of yeast Saccharomyces cerevisiae BZ34 used in these investigatious was kindly donated by Robert K. Mortimer and is described elsewhere 1~. This strain has non-complementing m u t a n t alleles in the arginosuccinase locus and requires arginine to be supplemented in the growth mediuin_. Induction of reversion to arginine independence after irradiation can be detected by plating the irradiated cells on a medium without arginine. Media

The nutritionally rich broth consisted of yeast extract, dextrose and peptone (YEPD). Nutritionally deficient (arginine-less) medium was prepared by using glucose, Yeast Nitrogen Base without amino acids and the required amino acids except arginine. This medium is designated Arg . Solid media for purposes of plating were prepared by adding 2% agar to the liquid media. Details of the media used were the same as described by MORTIMERet al. 1~.The culture was grown in Y E P D with constant shaking for 48 h at 3 o°. After the termination of growth the culture flasks were stored in a refrigerator at 5 ° till the time of use. Irradiation technic~uc

Investigations were carried out with both low and high L E T radiations. G a m m a rays from a 6°Co g a m m a cell and alpha rays from a ~l°Po electro-deposited silver disc served as low and high L E T radiations respectively. The g a m m a cell used had a dose rate of about 2.3 krad/min, and the alpha source had a dose rate of 21 rad/min. Cultures used during these experiments were harvested from the stationary phase. For g a m m a irradiation the cells were washed and resuspended in sterile distilled water to a final concentration of 5"1o7 cells/nil. Budding cell concentration in all

RADIATION-INDUCED REVERSION IN A YEAST STRAIN

18 5

s a m p l e s was less t h a n 5O/o. A b o u t 5-ml suspensions p l a c e d in screw-cap vials were e x p o s e d to a g a m m a dose of a b o u t 2 k r a d . The filter p a p e r t e c h n i q u e used for a l p h a i r r a d i a t i o n is described elsewhere n. S a m p l e s were e x p o s e d to an a l p h a dose of a b o u t i k r a d . Cell killing at these doses was negligible. Care was t a k e n to see t h a t the samples were m a i n t a i n e d at 5 ° before a n d after i r r a d i a t i o n till t h e t i m e of p o s t - i r r a d i a t i o n treatment. Post-irradiation treatment A f t e r i r r a d i a t i o n t h e ceils were r e s u s p e n d e d in 20 ml liquid Y E P D such t h a t t h e final c o n c e n t r a t i o n was a b o u t 2. 5 • IO 6 cells/ml. Samples were allowed to grow in r o o m light a t 3 °0 on a s h a k e r i n c u b a t o r . Samples were w i t h d r a w n at r e g u l a r intervals, w a s h e d t h o r o u g h l y to r e m o v e traces of n u t r i e n t b r o t h a n d t h e n r e s u s p e n d e d in distilled water. A p p r o p r i a t e dilutions of t h e same were done a n d p l a t e d on A r g - p l a t e s a n d Y E P D p l a t e s a n d i n c u b a t e d at 30 ° for a b o u t 3-5 days. Colonies were t h e n c o u n t e d a n d t h e reversion f r e q u e n c y was d e t e r m i n e d . Reversion frequency is expressed as t h e r a t i o of t h e n u m b e r of colonies on A r g - to Y E P D p l a t e s t i m e s the dilution factor. Cell counts a n d the b u d d i n g cell p e r c e n t a g e were also d e t e r m i n e d on a microscope at intervals. RESULTS AND DISCUSSION The v a r i a t i o n of reversion frequency w i t h t i m e of i n c u b a t i o n in liquid Y E P D m e d i u m is shown in Figs. I a n d 2 for garmlta a n d a l p h a r a d i a t i o n s respectively. The f r e q u e n c y increases r a p i d l y w i t h t i m e of i n c u b a t i o n , reaching a p e a k at a b o u t 9 ° min, a n d declines t h e r e a f t e r . A f t e r a p e r i o d of 8 h t h e reversion frequency a t t a i n s a c o n s t a n t v a l u e (see Fig. I). The p a t t e r n of v a r i a t i o n is similar for b o t h t y p e s of r a d i a t i o n . The r a t i o of t h e p e a k f r e q u e n c y to t h e zero t i m e f r e q u e n c y v a r i e d b e t w e e n 2 a n d 3 in separ a t e e x p e r i m e n t s . H o w e v e r , the positions of the p e a k a n d t h e p l a t e a u were reproducible for b o t h t y p e s of r a d i a t i o n . The initial rise is similar to t h a t o b s e r v e d in UV-irrad i a t e d b a c t e r i a 2°. However, in b a c t e r i a no decline in m u t a t i o n frequency was observed b y c o n t i n u i n g t h e i n c u b a t i o n in b r o t h as seen in these e x p e r i m e n t s . This is p r o b a b l y

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Fig. 2. Variation of reversion frequency with duration of incubation in broth after alpha irradiation (i krad). due to differences in the mechanisms of induction of the two types of genetic events-mutation and conversion. RAMON AND 3IORTII~IER13 have suggested that radiation induces single-strand breaks in DNA which act as precursors for the recombinational events. BrE~DEL AND HAYNES1 have shown that in yeast there are at least two independent pathways for the repair of DNA damage. One of these is called the 'X-repair' (similar to the excision-repair of UV-induced DNA damage) and the other the recombinational repair. In bacteria ~,9,~0, initiation of protein synthesis by incubating the cells in broth immediately after UV irradiation would inhibit the excision repair of DNA damage and fix the mutational lesions, resulting in an increased mutation frequency. Similar enhancement of UV-induced mutation yield has been demonstrated in haploid yeast 1~. The results of the present investigation can be explained on similar lines. \\'e can suppose that post-irradiation treatment in broth will immediately start macromolecular synthesis in the irradiated yeast cells inhibiting the 'X-repair' pathway thus making the lesions available for the recombinational pathway which results in an increased number of revertants. If one looks at the total number of cells per ml at various times during the post-irradiation growth (Fig. 3), it indeed appears that in the first 9° rain the cell number remains almost unchanged while the reversion frequency goes up by a tactor of 2 to 3. However, preliminary experiments of growing irradiated (2 krad) and unirradiated cells in both liquid Arg- and Y E P D media have shown that, in Arg- medium, only about x/a to 1/~ of the total number of cells can undergo the first division. The concentration of arginine in the intracellular pool of aminoacids may not be sufficient in all cells to support them to go through at least one division in an arginine-less medium. According to the copy choice model for heteroallelic recombination, post-irradiation DNA synthesis is a prerequisite for the expression of gene conversionl4, ~. This has been supported by the work of }~SPOSITO~where cells irradiated at advanced stages of DNA synthesis showed a decreased yield of recombinants. This would mean that, of the potential revertants, about 2/a to ~/z are suppressed from expression because the plating medium is unable to support them to go through the first post-irradiation DNA synthesis. However, this progression is easily achieved by incubating the cells in liquid Y E P D for some time. Hence it can he alternatively suggested that as the irra-

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diated cells are placed in the liquid Y E P D medium, it initiates the progression of cells in the division cycle more rapidly and extensively than the Arg- medium. At the lime when all the cells have reached the DNA synthesis stage the expression of gene conversion is maximal. In support of this hypothesis further experiments were performed. Since budding of yeast cells is a sure sign of progression in the DNA synthesis stateT, 18,19, the increase in budding cell population was determined microscopically at each interval of time aiter post-irradiation treatment in both Arg- and Y E P D media. This is shown in Fig. 3 : the budding cell population increased very rapidly up to about 9O~/o within the first few hours of treatment in YEPD. However, the peak value in the Arg- medium did not exceed 2O~/o. This shows that treatment in Y E P D medium after iriadiation can initiate DNA synthesis very rapidly. This result, along with the actual growth curve during the treatment, can oe used to explain the variation of ~eversion frequency with duration of post-irradiation treatment (Figs. I and 2). In the present Y E P D plating technique a viable mother-bud complex will form a single macro-colony on the plate. As the cells progress toward nuclear synthesis, while the chance t h a t a potential revertant being expressed increases, the total viable cell number, as determined by colony counts on the Y E P D plates, remains constant. Hence, the ratio of revertants per survivor increases with time. The time at which the peak occurs represents the minimal time required b y the cell population to reach a stage in the first post-irradiation division cycle at which the potential revertants can be expressed. Beyond this time, as seen from the growth curve, the mother-bud complex cleaves and the absolute cell number increases. Since in the

13. S. RAO el al.

1788

first division after the post-irradiation treatment only one of the daughters is a revertant while the other is not, at subsequent cell divisions the revertants per survivor decrease and reach a constant level. Because of the lack of synchrony in the cell population, the reversion frequency stabilizes only after a couple of divisions. To resolve the question whether or not the increase in reversion frequency obtained by treatment in broth is due to the inhibition of some non-recombinational repair pathways, thus making the lesions available for recombinational events, the following experiment was devised. The irradiated cells were first incubated for 9° min in liquid Arg- medium and then transferred to the nutrient broth. If the effect of the nutrient broth treatment in the earlier experiments was to inhibit non-recombinational repair pathways, then this regimen should result in the elimination ot the broth effect. However, as shown in Fig. 4, the broth effect is not lost suggesting that it is not due to creating an imbalance in the competitive repair pathways in favour of a recombinational mechanism. It may be noted that the ratio of the peak frequency to the zero time frequenc2 in this experiment is slightly less than that in Fig. I. This is probably because of the staggering effect of double treatment resulting in a wider spread in the progression of cells in the division cycle. Further experiments were done at high doses to determine the influence of division delay on the broth effect. In haploid yeast no modification in mutation frequency was observed after incubation in amino acid solution after high UV dose ~. This was taken to mean that the pre-mutational damage could be repaired during the division delay. However this does not appear to be true for intragenic recombination in diploid yeast. As seen in Fig. 5, even after a dose of 6o krad (lO% survival) inducing considmable division delay the broth effect was not lost suggesting that the lesions responsible for the stimulation of intragenic recombination are stable. In all these experiments the 7000

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Fig. 4. G a m m a irradiated cells (2 krad) were incubated in liquid A r g - m e d i u m for 9o nlin and t~ansferred to liquid Y E P D medium. Samples were w i t h d r a w n to score the reversion frequency after various incubation periods in Y E P D . Fig. 5. Variation of reversion frequency with d u l a t i o n of incubation in b r o t h after a high dose (6o krad) of g a m m a radiation.

RADIATION-INDUCED REVERSION IN A YEAST STRAIN

18 9

percentage increase of budding cells was closely in parallel with the increase in reversion frequency. These experiments further emphasize the intimate relationship between the expression of gene conversion and the synthesis of DNA during the first post-irradiation division cycle. ACKNOWLEDGEMENTS Thanks cal assistance

a r e d u e t o M. S. SIDHARTHAN AND V . V . D E O R U K H A K A R foY t h e i r t e c h n i in this work.

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