Radiation Botany, 1971, Vol. 11, pp. 483 to 485. Pergamon Press. Printed in Great Britain.
S T I M U L A T I O N OF T H Y M I N E U P T A K E I N T O CHLAMYDOMONAS FOLLOWING ULTRAVIOLET IRRADIATION A L A N R. I-IIPKI~S*
Biology Branch, Atomic Energy of Canada Ltd., Chalk River, Ontario, Canada
(Revised manuscriptreceived21 April 1971) HiPKiSS A. R. Stimulation of thymine uptake into Chlamydomonas following ultraviolet irradiation. RADIATIONBOTAN'¢I1, 483--485, 1971.--The effect of u.v. irradiation on the uptake of x*Cthymine into the nucleotide pool ofChlamydomonas reinhardihas been investigated. It was found that in the first hour after exposure to u.v. the ~'tC-thymine entered the cells at an increased rate. An apparent increase in the incorporation of x4C-thymine into nucleic acid was also detected. ~I'HE UNICELLULAR green alga Chlamydomonas reinhardi is used frequently in the study ofgenetic effects of ultraviolet (u.v.) and ionising irradiation(1,s,a) but little is known of the immediate metabolic effects following exposure of this organism to irradiation. Whilst investigating the immediate effects of u.v. irradiation on C. reinhardi, a stinmlation in thymine uptake into the cell was found to occur during the immediate post-irradiation period. Strain 11/32D of Chlamydomonas reinhardi was grown photosynthetically in liquid culture as previously described. (2) Logarithmically growing cells were harvested by centrifugation and resuspended in fresh growth medium at a concentration of about 6 × 10 ~ cells/ml. 25 ml of the cell suspension, in a 10 cm diameter Petri dish were exposed to 2537 A u.v. light delivered from a low pressure mercury vapour lamp at a rate of 42 ergs/mm2/sec. Following irradiation, the cell suspension was diluted with 25 ml of fi'esh growth medium containing 0"5 ~xCi of Me-14C',thymine (specific activity 3 [xCi/mM), illuminated and aerated, the final cell density being
around 3 × 106 cells/ml. At hourly intervals following exposure, 10 ml samples of the cells were taken which were then washed twice with distilled water, extracted with ice-cold 5 per cent trichloroacetic acid (TCA) for five rain, and finally centrifuged again, the supernatant fraction being designated the nucleotide pool. Radioactivity in the nucleotide pool was determined using a Packard liquid scintillation counter. Exposure to u.v. irradiation was found to give rise to an increase in the entry of 14C-thymine into the Chlamydomonas nucleotide pool (Fig. I). Exposure to u.v. is seen to stimulate the entry of thymine into the cell after overcoming a threshold level at 1260 ergs/mm 2, the effect increasing with the u.v. dose given. The effect appeared most marked following the shortest incubation period, which suggested it was transient. This was shown by determining the radioactivity in the nucleotide pool following incubation with l*C-thymine for five separate 30-min periods in the 3½ hr following irradiation. Table 1 shows that during the first and second
*Present address: Department of Biochemistry, University of London King's College, Strand, London, W.C.2, England. 483
between thymine and uracil in the u.v.-induced effect. Ultra-violet irradiation has been reported to damage the cell membranes of yeast cells resulting in leakage of 260 m F adsorbing substances (purine and pyrimidine bases, nucleosides and nucleotides) into the extracellular fluid.C5) The possibility of leakage from u.v.treated Chlamydomonas cells was therefore investigated during the first hour following exposure to 5040 ergs/mm 2 irradiation Leakage was indeed found to occur (Table 2) .This occurred
u . 300 o
, 2 ] n c u b o l ~ o n time,
Fro. 1. Uptake of 14C-thymine into the nucleotide pool ofC. reinhardi after exposure to 0 (O), 1260 (©), 2520 (A) and 5040 (fl) erg/mms u.v. irradiation.
Table 1. Effect of u.v. irradiation (5040 ergs/mm2) on uptake of 1*C-thymine into nucleotide pool of C. reinhardi
Incubation period, min after irradiation
Radioactivity in nucleotide pool, percentage of control unirradiated cells
0-30 30--60 60-90 120-150 180-210
213 186 84 81 90
30-rain periods the uptake of thymine was about twice that of the unirradiated control cells, while in the 30-min periods starting after one, two and three hr post-irradiation incubation, the uptake rate had decreased to control values or even less. The effect is not restricted to thymine as preliminary experiments indicate that the uptake of 14C-glycine, 14C-phenylalanine and 14Caspartic acid into the nucleotide pool in a onehr period immediately following 5040 ergs/mm 2 u.v. were 218, 198 and 195 per cent respectively of that for unirradiated cells. But no effect on liC-uracil uptake was found. No explanation can be offered for the apparent discrimination
Table 2. Effect of u.v. irradiation (5040 ergs/mm2) on leakage from C. reinhardi of 260 m~ adsorbing material into extracellularfluid
O.D. (260 mF) of extracellular fluid of: ~Jnirradiated Irradiated cells cells (sham irradiated) Before irradiation Immediately after irradiation 25 min after irradiation 50 rain after irradiation
during the irradiation period, after which time leakage ceased. It is possible therefore that damage to the Chlamydomonas cell m e m b r a n e occurs on exposure to u.v. irradiation which would give rise temporarily to both increased entry and exit of small molecules. It should be noted, however, that the u.v.-promoted leakage had ceased before the elevated thymine entry rate had disappeared which could argue against there being a direct correlation between the two effects. Alternatively it is possible to think of the increased thymine uptake as a secondary physiological response to the primary leakagepromoting u.v. effect. It should be pointed out that while survival determinations were not routinely carried out, it was found that greater than 70 per cent
SHORT COMMUNICATION survival was recorded following 5040 e r g / m m 2 irradiation. Furthermore, the possibility that both leakage a n d the stimulation of thymine entry were consequences of the 30 per cent cell death, a n d not results of m e m b r a n e damage, cannot be eliminated by the present data. However, it was found that when C. reinhardi was exposed to 20 k R X-irradiation (which gave greater than 90 per cent lethality(2)) no stimulation of thymine uptake was observed. This provides indirect evidence that the u.v.-promoted stimulation of thymine uptake was not a result of cell death. T h e effects on the entry into the cell of macromolecule precursors reported in this communication could complicate any investigation of post-irradiation macromolecule synthesis. For example Fig. 2a shows an a p p a r e n t increase in the incorporation of 14C-thymine into the cold T C A insoluble and hot T C A soluble fraction (nucleic acid) after exposure to 5040 ergs/mm ~ u.v. irradiation. Such an increase in incorporation, however, is most likely to result from the increase in the supply of radioactive thymidylate precursor to the nucleotide pool (Fig. 2b). This result argues against the possibility that the appearance of more radioactivity in the nucleotide pool of the u.v. irradiated cells is due to an inhibition of D N A synthesis causing an accumulation of D N A precursors. Acknowledgement--The author is grateful to the National Research Council of Canada for the award of a post-doctoral fellowship during the tenure of which the present investigation was carried out. REYERENCES 1. DAVIES D. R. (1965) Repair mechanisms and variations in u.v. sensitivity within the cell cycle. Mutation Res. 2, 477--486. 2. Hxprass A. R. (1967) Immediate loss of flagella from Chlamydomonas reinhardi after ultraviolet irradiation. Radiation Botany 7, 347-349. 3. LAWRENCEC. W. (1967) Influence of non-lethal doses of radiation on allelic recombination in Chlamydomonas reinhardi. Genet. Res. 9, 123-127. 4. POSNERH. B. and SPARROWA. H. (1964) Survival of Chlorella and Chlamydomonas after acute and
chronic gamma radiation. Radiation Botany 4, 253-257. 5. SmOEL S. J. and SWENSONP. A. (1964) Loss of nucleotide and amino acid pool components from yeast cells following exposure to ultraviolet and photoreactivating radiations. 07. Cell. Comp. Physiol. 63, 253-260.
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FIo. 2. Effect of 5040 u.v. on (a) the incorporation of
1*C-thymine into C. reinhardi nucleic acid (cold TCA insoluble, hot TCA soluble) and (b) the uptake of 14C-thymine into the nucleotide pool of C. reinhardi. O = u.v. irradiated cells. • = control unirradiated cells.