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MASTER
RWATION OF THE STUDIOS ON MUTAGENESIS IN PARAMECIUM TO THE
DOSE RATE PROBLEM*
R. F. Kimball
Biology Division, Oak Ridge National Laboratory,
Oak Ridge, Tennessee
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*Research sponsored by the U. S. Atomic Energy Commission under contract
with the Union Carbide Corporation.
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3.
INTRODUCTION
The influence of dose rate and dogo fractionation upon the yield of
mutations can have a nu ser of different explanation as earlier papers
in this conference have pointed out. The explanation for one class of
mitational change, two-hit chromosomal aberrations, as Dr. Wolff pointed
out in his paper is especially simple and direct. The two breaks that are
required for such aberrations must be produced close enough to each other
&..
.
.
in space and time to interact. Otherwise the breaks restitute. Consequently
Interaction will be incomplete if the total dose 18 given over a period of
time that is long compared to the mean time for restitution.
Evidence also exists suggesting that the yield of one-hit mutational
events can be influenced by the time over which the dose is given (Russell,
Russell, and Kelly, 1960; Sobels, 1963; Tazima et al., 1961; Tazima and
Kondo, 1963), but no comparably simple and direct explanation exists. Most
of the explanations have used the idea that result from side effects of the
radiation on repair mechanisms, on the duration of the cell cycle, or on
cell survival. A side effect on repair mechanisms requies that one-hit
mutations arise from initially reparable premutational damage; side effects
on the cell cycle and on cell survival require differences in mutation yield
at different stages of the cell cycle or in different cell types. Our
studies with Paramecium aurelia provide unequivocal evidence for both these
.
E'
2.
requirements. One of the purposes of this paper is to review this evidence.
Another is to show why it is that despite these features there is in
Paramecium no dose rate or dose fractionation effect for one-hit mutations
induced by X rays.
METHODS
The method for inducing and detecting mutations in P. aurelia has been
described a number of times (Kimball and Perdue, 1963). Essentially it 18
as' followe. Synchronized groups of paramecia are obtained by picking dividing
specimens out of 108 phase cultures and irradiating at a known time after
division. Single cell isolations are made after irradiation, and the isolates
are allowed to multiply by cell division to form small cultures. When the
food supply in these cultures is exhausted, autogamy, a sexual. process that
produces complete homozygosis, occurs. From each culture, 25 autogumous
specimens are isolated, and the frequency of these isolates that die or
grow poorly is determined. This frequency is converted to a quantity, M,
that equalizes the variance over a wide range of values, thus making statistical
analysis easier.
TWO-HIT VERSUS ONE-HIT MUTATIONS
Both two-hit chromosomal aberrations and one-hit aberrations or point
mitations could produce lethal and sublethal segregants at autogamy. No
direct 'test to distinguish between these two classes has yet been devised
for P. aurelia, but dose rate and dose fractionation studies provide an
indirect test since two-hit aberrations should be eliminated by sufHciently
increasing the time over which the dose is given. It is, of course, possible
that one-hit mutations will also be reduced, but a maximum estimate of the
contribution of two-hit aberrations is obtained by attributing the whole
dose rate effect to this class.
The data indicate (Kimball, 1963b) that the contribution of two-hit
aberrations must be small. For example, decreasing the dose rate to such
an extent that almost the whole of the Gl period is required to give the
dose reduces the yield of inviable and poorly growing autogamous segregants
by no more than 20 per cent (Figure 1). The curve in Figure 1 suggests that
the half life for the breaks Involved in two-hit aberrations must be no more
than a few minutes. Tosts woro made for breaks with much longer half lives
by giving the dose in two equal fractions in the early Gl period of successive
cell cycles on the assumption that breaks could not remain rejoinable any
longer than one cell cycle. No evidence for breaks with long half lives was
found.
Indeed at the dose rate used, it was impossible in a series of 13
experiments to demonstrate a statistically significant effect of fractionation.
The small difference actually found was in the right direction and of about
the size anticipated f:om the dose rate experiments.
It must be concluded from the dose rate experiments that the maximum
contribution of two-hit aberrations to postautogamous lethality and slow
growth 18 20 per cent and that the remaining 80 per cent results from one-hit
mutations. The rest of the paper deals with the properties of these one-hit
mutations.
PREMUTATIONAL DAMAGE AND REPAIR
At the International Symposia in Genetics at Tokyo in 1956, I (Kimball,
1957) first reported that several quite different treatments can decrease
the mitation yield when they are given after X irradiation. Modification of
the mutation process itself, not artifacts of selection or alterations or
gene expression, were shown to be responsible for this effect (Kimball,
Gaither, and Wilson, 1957). The various treatments had little in compon
except that they delayed division and presumably chromosome replication;
consequently it was hypothesized that they all provided more time for a
spontaneous repair process that ended when the chromosomes replicated.
Evidence was presented in these and later papers (see Kimball, 1963a, for
review) that the longer the time to DNA synthesis the less the mutation.
metabolizing cells. This finding suggests a metabolically controlled
repair system, and, taken in conjunction with evidence for DNA repairing
enzymes (Harm, 1963; Rupert, 1964; Setlow and Carrier, 1964; Boyce and
Howard-Flanders, 1964), led to the view that X rays produce lesions in the
chromosomes that can be repaired enzymatically until the time of DNA synthesis
when the remaining lesions are converted to final mutation (see Kimball, in
press, for further discussion of the nature of these lesions).
It has been suggested that mutations also could arise by errors in
repair processes (Setlow, 1964). In Paramecium, a premutational lesion
must have a greater chance to produce a mutation by remaining unrepaired
until replication than it does by undergoing an error in repair. This
conclusion follows from the fact that the maximum number of mutations is
produced by irradiating just before DNA synthesis when the chance of repair
before replication 18 minimal. This would be an expected consequence, of
course, if all lesions that are unrepaired at the time of replication were
converted to mutation; but no evidence is available about the efficiency
of conversion to mutation at replication other than that it is more efficient
thara conversion during "repair." Actually, there is no critical evidence for
this latter process in Paramecium. However, the yield of inviable and
poorly growing autogamous segregants approaches a lower limit of about
one-quarter to one-third its maximum value as the time to DIVA synthecis
becomes very long (Kimball, 1963a), and this lower limit could result from
mutations produced by errors in repair.
.
THE G2 AND EARLY PROPHASE PERIOD
The previous sections dealt entirely with cells Irradiated in G1.
X irradiation in G2 or early prophase produces no more than one-tenth the
mutation produced by irradiating in G1 (Kimball and Perdue, 1963). The
evidence suggests, though it does not prove, trat this low yield is a
consequence of extremely efficient repair of premutational damage, though
there is no a priori reason why repair should be so efficient during these
particular parts of the cell cycle. From the present point of view, the
low yield during G2 and early prophase is important because (1) it shows
that a very large fraction of all one-hit mutations, possibly all, rise
from initially reparable premutational damage, and (2) it shows that large
variations in the effectiveness of X rays for producing one-hit mutations
occur during a single cell cycle.
OTHER MUTAGENS, THE GENERALITY OF REPAIR PROCESSES
Similar evidence for reparable premutational lesions has been obtained
with other mutagens. Posttreatment with streptomycin (Kimball, Gaither,
and Wilson, 1959) decreases the amount of mutation produced by X rays but
also the amount produced by 2537 A ultraviolet light, Pu 9 alpha particles,
and the alkylating agents, nitrogen mustard and triethylenemelanine (Table 1).
These same mutagens have all been shown to produce appreciably less mutation
in G2 than in G1, though the ratio ranges from greater than 10 for X rays
to about 3 for triethyleneme lamine. Except for alpha particles, which have
not been tested, mutagenic treatments given just before DNA synthesis produce
the maximum amount of mutation with lesser amounts produced the earlier in
Gl the treatment is given. These results suggest that the diverse premtagenic
lesions produced by these mutagens are all subject to repair until the
time of DNA synthesis when the remaining lesions are converted to final
mutation. The general conclusion 18 that repair of premutational lesions
1.8 a general error correcting mechanism, not just the property of one or
a few kinds of initial lesions (cf. Patrick et al., 1964).
The one exception to the generality of repair mechanisms in Paramecium
and in other organisms as well is photoreversal. In Paramecium, as in
other organisms, the yield of mutations from short wavelength (about 2600 A)
ultraviolet light can be considerably decreased by subsequent exposure to
long wavelength (about 3600 A) ultraviolet and short visible light, but no
comparable effect is found with X-ray induced mutations (Kimball and Gaither,
1951). Witkin (1964) has presented evidence for ultraviolet-induced
prototrophic reversions in Escherichia coli that the photoreversing light
acts indirectly by promoting dark repair. She finds evidence, however, that
photoreversing light prevents the production of ultraviolet-induced mutations
to streptomycin resistance by splitting thymine dimers. The data for lethal
and slow growth mutations in Paramecium are more in accord with this latter
method of action since thymine dimers are only produced by ultraviolet
light whereas dark repair systems seem to be general for all mutagens. If
photoreversing light were acting by modifying the dark repair system, it
should be effective with X rays as well as with ultraviolet.
· DOSE RATE AND DOSE FRACTIONATION EFFECTS WITH ONE-HIT MUTATIONS
I now turn to the reason why dose rate and dose fractionation effects
are not found for one-hit mitations in Paramecium. To answer this question
it is necessary to summarize the explanations that have been offered for
other organisme. In the mouse (Russell, Russell, and Kelly, 1960; Pus ell,
1963), two alternative explanations for the dose rate effect have been
suggested involving reparable premutational damage: (1) The repair system
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can be saturated it too many lesions are produced at one tirie. (2) The
repair system can be damaged by the radiation itsolr by it doen.rnte dependent
process. Tazima and Kondo (1963) seem to favor the latter interpretation
for the type I dose rate effect in the silkworm. There is no obvious
reason why the repair system in Paramecium would be less readily saturated
than that in the mouse, but there is good reason to think that the repair
system would be less readily damaged by X rays. Paranecium, like other
ciliates, is very resistant to the division-delaying action of x rays and
probably to various metabolic effects as well. Thus a repair system
involving some part of cellular metabolism would probably have very little
chance of being damaged at the relatively low (for Paramecium) doses of
X rays needed to produce mutation. Consequently, the difference between
the mouse and Paramecium 18 expected on Russell's second hypothesis. The
Paramecium data give additional support to this hypothesis by showing that
many one-hit mutations if not all arise from reparable premutational damage.
Dose fractionation effects in the mouse (Russell, 1962; 1963) and type II
dose rate effects in the silkworm (Tazima, Kondo, and Sudo, 1961; Tazima
and Kondo, 1963) have been quite a different set of interpretations involving
differential cell killing, partial synchronization of the cell cycle, or
some combination of these two. All these explanations depend on the idea
that there are appreciable differences in sensitivity to mutation induction
during the cell cycle or among cells of different types. The Paramecium
data show that this can be true for one-hit mutations. The obvious reason
.
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20
why dose fractionation effects do not occur in Paramecium 18 that mutagenic
doses of X rays neither kill this species nor appreciably alter its cell
cycle. Moreover, we have been able in the dose fractionation studies to
give each fraction at the same stage of the cell cycle, thus avoiding the
effect of any small altoration of the cell cyclo that may have occurred.
FRACTIONATION EXPERIMENTS WITH ULTRAVIOLET LIGHT
Some idea of the consequences for mutation yield in Paramecium of
using a mutagen that markedly affects the cell cycle can be obtained from
a few preliminary experiments on dose fractionation with 2537 A ultraviolet
light. Mutagenic doses of ultraviolet light not only greatly prolong the
irradiated cell. generation but several later cell generations as well
(Kimball, Geckler, and Gaither, 1952). Consequently even irradiating at
a known time after division 18 insufficient to standardize conditions since
the time between irradiation and DNA synthesis will vary for each fraction
depending on the previous radiation history. Moreover, cellular metabolism
and repair processes are probably not the same at different times after
the beginning of the series of fractional doses.
The results in Table 2 bear this out, at least in a general way. In
two experiments in which the fractions were given in different cell cycles
but with different fractionation schedules, fairly large and statistically
significant effects of fractionation were found, though in the different
directions. We are reminded of the mouse work (Russell, 1963) in which
quite different results were found depending on the fractionation schedule.
Even greater effects would have been found if we had not controlled the
time after division at which the successive fractions were given. In two
other experiments, in which a series of 10 fractions were given within a
single Gl period with short intervals between the fractions a statistically
significant decrease over the single-dose controls was found in one
experiment; no effect in the other.
In the positive experiment, the
fractionated dose group showed a markedly greater division delay than the
single-dose group; in the nocative experiment, there was no difference in
division dolay between the two groups. The variability of the results
shows that we have less control over the ultraviolet experiments than would
be desirable, but the obvious relation to the effect on the cell cycle adds
support to the idea that radiation-induced modifications of the cell cycle
and probably of cell metabolism are fundamental for dose rate and dose
fractionation effects on one-hit mutations.
SUMMARY
Mutation studies with Paramecium aurelia have demonstrated that most
if not all mutations arise from initially reparable premutational damage.
This generalization seems to hold for such diverse mutagens as X rays,
alpha particles, ultraviolet light, and alkylating agents. These studies
also demonstrate that large variations in the yield of one-hit mutations
occur during the cell cycle. Despite these two features, there is little
18 any dose rate and dose fractionation effect with X-ray induced, one-hit
mutations. The reason for this seems to be that mutagenic doses of X rays
have almost no effect on the cell cycle and no effect on preautogamous
survival of this organism. Thus the effects of radiation on repair mechanisms,
on the cell cycle, and on cell survival, which have been used to account for
dose rate and dose fractionation effects in other organisms, are missing in
Paramecium. Mutagenic doses of ultraviolet light do affect the cell cycle
in Paramecium, and, in keeping with expectation, fractionation of the
ultraviolet dose has clear effects on mutation yield.
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LITERATURE CITAD
Boyce, R. P. and P. Howard-Flanders 1964 Release of ultraviolet
Light-induced thymine dimers from DNA in E. coli K-12. Proc. Natl.
Acad. Sci. U.S. 51: 293-300.
Harm, W. 1963 Repair of lethal ultraviolet damage in phage DNA. In,
Repair from Genetic Radiation Damage, ed., F. H. Sobels, Pergamon
Press, Oxford, pp. 107-124.
Kimball, R. F. 1957 Modification of the genetic effects of X rays by
treatment after irradiation. Cytologia, supplement, 252-255.
Kimball, R. F. 1963a The relation of repair to differential radiosensitivity
in the production of mutations in Paramecium. In, Repair from Genetic
Radiation Damage, ed., F. H. Sobels, Pergamon Press, Oxford, pp. 167-176.
Kimball, R. F. 19630 X-ray dose rate and dose fractionation studies on
mutation in Paramecium. Genetics 48: 581-595.
Kimball, R. P. in press Studies on radiation mitagenesis in microorganisms.
Proc. XI Internatl. Congr. Genetics, vol. 2, Pergamon Press, Oxford,
1
11
Kimball, R. F. and Nenita Gaither 1951 The influence of light upon the
action of ultraviolet on Paramecium aurelia. J. Cell. Comp. Physiol.
37: 211-233.
Kimball, R. F., Nenita Gaither, and Stella M. Wilson 1957 Postirradiation
mods.fication of mutagenesis in Paramecium by streptomycin. Genetics
42: 661-669.
Kimball, R. F., Nenita Gaither, and Stella M. Wilson 1959 Reduction of
mutation by postirradiation treatment after ultraviolet and various
kinds of ionizing radiations. Radiation Research 10: 490-497.
Kimball, R. F., R. P. Geckler, and Nenita Gaither 1952 Division delay by
radiation and nitrogen mustard in Paramecium. J. Cell. Comp. Physiol.
40: 427-460.
Kimball, R. F. and Stella W. Perdue 1963 Studies on the refractory period
for the induction of recessive lethal mutations by X rays in Paramecium.
Genetics 47: 1595-1607.
Patrick, M. H., R. H. Haynes, and R. B. Uretz 1964 Dark recovery phenomena
in yeast. I. Comparative effects with various inactivating agents.
Mutation Research 21: 144-163.
Rupert, c. S. 1964 Photoreactivation of ultraviolet damage. In,
Photophysiology, vol. II, ed., A. C. Giese, Academic Press, New York,
pp. 283-327.
Russell, W. L. 1962 An augmenting effect of dose fractionation on raviation-
induced mutation rate in mice. Proc. Natl. Acad. Sci. U.S. 48: 1724-1727.
Russell, W. L. 1963 The effect of radiation dose rate and fractionation on
mutation in mice. In, Repair from Genetic Radiation Damage, ed.,
F. H. Sobels, Pergamon Press, Oxford, pp. 205-217.
Russell, W. L., Liane B. Russell, and E. M. Kelly 1960 Dependence of
mutation rate on radiation intensity. Internat. J. Radiation Biol.
Suppl. 311-320.
Setlow, R. B. 1964 Physical changes and mutagenesis. J. Cell. Comp. Physiol.
: Suppl.
Setlow, R. B. and W. L. Carrier 1964 me disappearance of thymine dimers
from DNA: an error-correcting mechanism. Proc. Natl. Acad. Sci.
51: 226-231.
Sobels, F. H. 1963 Repair and differential radiosensitivity in developing
germ cells of Drosophila males. In, Repair from Genetic Radiation
Damage, ed., F. H. Sobels, Pergamon Press, Oxford, pp. 179-197.
Tazima, Y. and S. Kondo 1963 Differential radiation-sensitivity of germ
cells as a possible interpretation of sex difference in dose-rate
dependence of induced mutation rates in the silkworm. In, Repair from
Genetic Radiation Damage, ed., F. H. Sobels, Pergamon Press, Oxford,
pp. 237-248.
Tazima, Y., S. Kondo, and T. Sado 1961 Two types of dose-rate dependance
of radiation induced mutation rates in spermatogonia and oögonia of the
silkworm. Genetics 46: 1335-1345.
Witkin, E. M. 1964 Protoreversal and "dark repair" of mutations to
prototrophy induced by ultraviolet light in photoreactivable and
non-photoreactivable strains of Escherichia coli. Mutation Research
1: 22-36.
&
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15
BATE
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Table 1
Effect of Posttreatment with Streptomycin on Mutation
Induction in Parameciwa
Mutagen
Mutation (M Units)
Ratio
No Streptomycin
Streptomycin
X rays
0.72
0.66
a particles
2537 A UV
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0.99 + 0.08
1.17 0.08
1.01 + 0.06
1.23 t 0.05
0.84 1 0.06
0.66 $ 0.08
0.64 + 0.07
0.53 + 0.05
0.93 $ 0.05
TEM
0.52
HN2
0.76
Table 2
Effect of Dose Fractionation on Mutation Induction by about
2000 ergs/mm, Incident Intensity of 2537 A ultraviolet Light
No. of
Interval between
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