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MICROCOPY RESOLUTION TEST CHART
NATIONAL BUREAU OF STANDARDS - 1963
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ORN V 3212 NAATID
CONF-%70632--2 MASTER 28
FAUG 25 1967
To be presented at The Brookhaven
Symposium on "Recovery and Repair
· Mechanisms in Radiobiology" June
:
5-7, 19898 in Recovery Brookhar
CESTI PRICES
mm
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HC $ 3.0d, MN 65
DOSE-RATE EFFECTS ON INACTIVATION AND MUTATION-INDUCTION
IN NEUROSPORA CRASSA* :
F. J. de Serres, H. V. Malling and B. B. Webber
Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee
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with the Union Carbide Corporation:
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LEGAL NOTICE
This report was prepared ao an account of Government sponsored work. Neither the United
States, nor the Commission, nor any person acting on behalf of the Commission:
A. Makes any warranty or representation, expressed or implied, with respect to the accu-
racy, completeness, or usefulness of the information contained in this report, or that the uso
of any information, apparatus, method, or proceso disclosed in this report may not infringe
privately owned rights; or
B. Assumes any liabilities with respect to the use of, or for damages resulting from the
use of any information, apparatus, method, or procese disclosed in this report.
As used in the abovo, "person acting on beball of the Commission" Includes any on-
ployee or contractor of the Commission, or employeo of such contractor, to the extent that
guch employee or contractor of the Commission, or employee of such contractor prepares,
disseminates, or provides acceso to, any information pursuant to his employment or contract
with the Commiasion, or his employment with such contractor,
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*PERIODUTOR OF THIS DOCUMENT IS UNLIMITED
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- . I. INTRODUCTION
The very elegant studies on bacteria and viruses reviewed by Dr.
Witkin ) and Dr. Setlow have revealed an amazing array or complexity
in these organisms and they certainly represent major steps forward iri"
our attempts to obtain an understanding of the mechanisms of inactivation,
mutagenesis, and recovery, after exposure to radiation, at the molecular
level. It is clear that the primary target for inactivation and mutation-
induction in these organisms is DNA that there are different types of".
lesions ranging from alteration of a single base to double strand breaks
and that there are a variety of repair mechanisms. s
One cannot help but wonder, however, to what extent these findings
apply to higher diploid organisms (aukaryotes) where the DNA is organized
into chromosones with a more complex chemical structure and where these 1:
chromosomes are located in cells with often highiy specialized function.
Although many models of chromosome structure have been proposed for diploid
organisms we still do not know the precise organization. Even the relatively
simple question of the fate of a double-strand break in DNA cannot be
answered on theoretical grounds. What for example, is the fate of such
a lesion? Does it result in chromosome breakage in higher organisms?
Or is it perhaps just an intralocal deletion of some gene?
Even in the absence of any of this essential information on the
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answers to many of the same questions posed by many investigators in
experiments with viruses and bacteria.
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What are the most important 'targets?
What type of lesions are produced?"
How are these lesions repaired: 1
How does the initial lesion produce inactivation or mutation?
Is there a qualitative difference betweeri ºlethal and mutagenic damage?
II. The development of a eukaryote assay system
For a number of years we have been trying to develop. & microbial
288ay syatem with a eukaryote to study radiation-induced inactivation
and mutation-induction as well as the recovery from this damage at the
molecular level. Until fairly recentiy, the primary effort has been
placed on the characterization of radiation-induced genetic damage
resulting in inactivation and the induction of recessive lethal mutations
at specific loci. We have concentrated on the characteristics of X-ray-
induced genetic damage. In these studiesBot, we have found that the
same spectrum of genetic alterations' resulting in recessive lethal
mutation at specific loci can be obtai:nd with this new assay system as
is found with higher diploid assay systems. The basic features of our
system make it possible to study induction and recovery phenomena at
specific loci.both quantitatively and qualitatively in a manner that 18
egsentially impossible in higher organisms. The characterization of
the recessive lethal mutations at specific loci, by a variety of genetic
tests has shown that they result both (1) from point mutation and (2)
from gene loss by multilocus deletion. The spectrum of x-ray-induced
genetic alterations resulting in point mutation have been identified at
the molecular level by means of tests for specific revertibility after
treatment with selected chemical. mitagens The mutations due to
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gene loss have been shown to result from a series of overlapping chromosome
deletions that oftern include other known gene loci in the immediate
adjacent regions.
Thus, it appears that the genetic damage detectable with this new :
assay system includes all possible types and ranges from that which results
in mutation by a single base-pair substitution to that, which results in
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mutation by chromosome breakage.
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. In the present experiments, we have varied the recovery conditions |
to determine whether these different types of genetic demage are selectively
repaired. .
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III. Characteristics of a two-component heterokaryon of Neurospora
The assay system consists of a two-component heterokaryon of Neurospora
crassa, a haplóid organism with no permanent diploid nucleus. . We have
been able to mimic a diploid system by placing two different, genetically :
marked, haploid nuclei together in the same cytoplasm.. This particular
arrangement has many advantages over the normal diploid condition partidularly
with regard to the genetic analysis of the recessive lethal mutations.
Two-component heterokaryons form 3 different types of asexual spores or
conidia. When the nuclear ratios are about equal, 15 to 25% of the conidia
are heterokaryotic, that is to say that they contain at least one nucleus :
of each genotype. Owing to the presence of biochemical markers in each
of the two components only the heterokaryotic fraction will grow on
minimal medium.
The genotype of each component of the heterokaryon is shown in Table
The heterokaryon 18 heterozygous for two closely linked genes (ad-3A and
ad-3) which control separate and sequential steps in purine biosynthesis.
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Mutation of either gene results in a requirement for adenine as well as
accumulation of a reddish-purple pigment in the vacuoles of the wycélium.
To recover the recessive lethal mutations induced at these two loci in
the haterokaryotic conidia, a low level of adenine is added to the basal.
medium. In this way only the unmitated heterokaryotic conidia and those
containing recessive lethal mutations in the ad-3 region will form colonies.
Homokaryotic conidia of either genotype do not have the essential growth
factor requirements and die rapialy in this medium. .:.
We have developed & direct methods to recover mutants baseð.
on pigment accumulation rather than by their requirement for adenine..
Because of this the moutant sample is unbiased and the purple colonies
recovered range from those which grow at wild-type rate on minimal medium
to those with complete requirements for adenine. With this direct method
untreated or treated samples of conidia are inoculated into 10 liters
of medium in 12 liter Florence flasks. These flasks are incubated in
the dark at 30°C for a period of 7 days.. Flasks are inoculated so that
there will be, on the average, 1 x 10° heterokaryotic survivors per flask.:
111 of the heterokaryotic survivors form colonies, about 2 mm in diameter,
during this growth perioi. The flasks are harvested by measuring the o
volume of each of a series of samples which are then poured into white
photographic developing trays. 81X-10 ml samples of background colonies,
are reserved for direct counts (to determine the total number of:
heterokaryotic survivors per flask) and the reddish-purple mutants
are then isolated by hand. Survival curves result from the relation
between the background colony counts of untreated and treated series.
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The frequency of the reddish-purple colonies among the totai colonies in
each flesk in control and treated series are used to develop dose-effect
curves for mutation-induction. i :
IV. Characteristics of the dose-effect curves for X-ray-induced inactivation
and mutation induction
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.: The characteristics of the dose-effect curves for X-ray-induced ***
inactivation and mutation-induction were first determined in experiments
with" X-rays at ice-water temperature In these experiments, simple,
exponential survival curves were found for the heterokaryotic conidia.
(Figure 1). 'We imow from these, as well as other experiments that :
inactivation results from some single event in one of the two nuclei in
a heterokaryotic conidium. This event converts the heterokaryotic
: conidium, which would ordinarily grow on minimal medium, to a homokaryotic
.conidium with the requirements imposed by the biochemical markers in one
of the haploid components. This single event has not been identified,
; and the mechanism of nuclear inactivation of heterokaryotic conidia 18"
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The dose-effect curve for the induction of all types of recessive
lethal mutations at the two loci in the ad-3 region has a slope of about
| 1.36 on a log-log plot' (Figure 2). By genetic analysis of the recessive
lethal mutations we have shown that this curve is a composite of
many different events and has two main components: point mutations
(ad-3) which increase linearly with dose and multilocus deletions
(ad-3") which increase as the square of the dose (Figure 3).
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One of the particularly interesting aspects of these data is that
they show that the spectrum of recessive lethal mutation 18 dose-dependent.
At low doses, Essentially all mutants result from point mutation and the
dose-effect curve will have a slope of about 1.0. At very high doses :
the recessive lethal mutations will be a mixture of point mutations and
chromosome deletion and the dose-effect curve will increase as some power
of the dose between 1.0 and 2.0...
In review, recessive lethal' mutations induced at the ad-3A and ad-3B
loci and recovered in a heterokaryon consist of (1) point mutations -
these result from single events within each locus producing mutants of
genotype ad-3A" and ad-3BR, and (2) multilocus deletions - these result
from single events which cooperate in pairs to produce mutations by gene
2088 of either locus or both loci simultaneously. "Such mutations are of
genotype ad-3A, ad-3BIR, and (ad-3A ad-3B)IR:
V. The nature of the lesions that result in inactivation and the induction
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2.
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The genetic analysis' of the recessive lethal mutations in the ad-3 |
region has shown that the single events that result in point mutations
are detected at a frequency 74 times lower (JFrequency of ad-3-R mutations/
frequency of ad-3* mutations) than the frequency of the single events
that cooperate in pairs to produce mutation by chromosome deletion. There
is a quantitative difference between these two events. But this is
probably due to the fact that the target for the latter events is much
larger and includes not only the ad-3 but also the immediately adjacent
genetic regions. But the important question is do these mutations result
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from lesions that are qualitatively different? Do the same lesions that
occur within the ad-3A and ad-38 locus to produce point mutations cooperat
:: in pairs to produce mutations by multilocus deletion? .
3. The genetic analysis of the recessive lethal mutation has also
showzí that point mutations and multilocus "deletions have different modes
of origin. If all X-ray-induced lesions are qualitatively identical one
would expect point mutations to be converted to multilocus deletions by
additional hits in one of the immediately adjacent régions. That is to i
.::. say that at high doses there would be en interaction between the events :
inside the locus (that would normally produce point mutations) and
events occurring outside of the locus that would, effectively, convert:
point mutations to multilocus deletions. If this were true, the frequency : ", i uhe's
of point mutations at high doses should increase as some power of the
: dose less than 1.0. Although there is a general decline in the forward-
mutation frequency at the 40 KR dose (Figure 2), the decline in the .
frequency of point mutations is not greater than the decline in the
frequency of multilocus deletions (Figure 3): Thus these data provided
strong support for the hypothesis that the lesions that produce multilocus
deletion are qualitatively different from the lesions that produce point:
mutation.
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VI. Dose-rate effects on heterokaryons of Neurospora
If the induction frequencies of the lesions resulting in point ::
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differentially under conditions of repair, we can conclude that the two
types of lesions are qualitatively different..
: It is well known that the frequency of 2-hit chromosome aberrations
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can be altered by changing the rate of radiation exposure. . When high"
intensity exposures are made, mary chromosome breaks are produced
simultaneously and they interact through misrepair to form a variety nf
chromosome aberrations. When the same exposures are given at low intensity
the yield of chromosome aberrations decreases because some of the breaks
are repaired before the remainder are produced thus lowering or eliminating
the opportunity for interaction. In this way we hoped to demonstrate
repair of the lesions that produced mutation by multilocus deletion and, in
perhaps , no repair of the lesions that produce point mutation.
The effect of dose-rate was studied in experiments where the rate
of exposure was reduced from 1000 R/min to 10 R/min. Thoroughly filtered
coñídial suspension (in 1/15M phosphate buffer, pH 7.0) of the two-
component heterokaryon described in Table 1 were collected on Millipore
i filters, and maintained on the surface of filter paper (noistened with
1/15M phosphate buffer, pH 7.0) in petri plates, prior to, during, and
following X-ray exposures of 5, 10, 20 and 40 kr. Since repair is known :
to be enzymic the irradiation was performed at a room temperature of 27°C.
- Since approximately 66 hours were required to deliver the 40 kr exposure
with the lower dose rate of 10 R/rain, a comparison was made between
1000 R/min exposures where conidia were inoculated into medium immediately,
with those where inoculation was delayed for 66 hours after Irradiation.
This 66 hr. delay provided evidence for a totally unexpected and striking
nonchromosomal dose-rate effect. .
Nonchromosome..) dose-rate effects on colony morphology ..
Untreated heterokaryotic conidia usually form perfectly sphericaz
colonies about 2 mm in diameter under the incubation conditions described
above. With high Intensity X-ray exposures of 1000 R/min, some of the
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colonies formed by the heterokaryotic survivors have an abnormally
Irregular morphology; they range in size from 2 mm in diameter down to :
those which are barely visible macroscopically. The percentage of abnormal
colonies among the total survivors is dependent on the total radiation
exposure. This is a well known effect of radiation ar.d has been repeated
many times in different laboratories. Delay of the inoculation for 66'.
hrs. resuits in a drastic reversal of this effect. There is a dramatic
shift in morphology from abnormal to the normal morphology found with
untreated conidia. The restoration of normal morphology is attributed
to recuvery processes which repair X-ray-induced damage during the post-
Irradiation storage period with the high intensity exposures. An equally
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2 It is our opinion that this repair is nonchromosomal; that it is
repair at the cytoplasmic level, because, as we will see in the later *
.. sections, there are only minor differences between the dose-effect curves
for the two different high intensity exposures. We believe that these
minor differences are due to relative difficulties in determining the
total number of colonies and the total number of ad-3 mutant colonies
accurately when abnormal morphology is encountered.
::. For this reason, we believe that the most valid comparisons in
these experiments are those between the dose-effect curves for the 2018 :
R/min exposures and those 1000 R/min exposures followed by 66 hrs post.
irradiation storage.
Dose-rate effects on inactivation of heterokaryotic conidia .
: A comparison of the dose-effect curves for survival of the
heterokaryotic conidia exposed at 27°C with the survival curve.obtained
from earlier experimento at 4°c (Figure 1) shows (1) that the final slopes
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of the 1000 R/mi:a curves at room temperature are not significantly different:
from the slope of the curve obtained at ice-water temperature, (2) that
there is little or no effect of post-irradiation storage at room temperature,
; and (3) that both of the survival curves at room temperature have a small
"shoulder" which is not found at ice-water temperature.
.:With exposures at 10 R/min much higher levels of survival were:
obtained, and the final slope of the curve is significantly different
from the 1000 R/min curves. The higher levels of survival obtained with 1.0
R/min can be attributed to repair of damage at room temperature that is in
lethal with 1000 R/min exposures. Repair of this damage is dose-rate
dependent since much hi.gher levels of survival were obtained at the lower
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Dose-rate effects on the frequency of recessive lethal mutations resulting from
genetic alterations in the ad-3 region
The dose-rate experiment was planned to cover that part of the
mutation-induction curve where the maximum difference was expected between
the overall forward-mutation frequencies as well as between the frequencies
of that class of recessive lethal mutation resulting from 2-hit multilocus
deletions. The overall forward: mutation curves that we obtained with the
1000 R/min exposures at room temperature (Figure 5) shows that there is a
marked saturation at the highest exposures which was not expected on the
basis of our earlier experiments performed at ice-water temperature. This
saturation has resulted in a considerable departure from the forward-
mutation frequencies expected with the 20 and 40 KR exposures at room
temperature. It is clear, however, that lowering the dose-rate to
2.0 R/min gave a significant reduction from the slope of 1.36 expected.
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The dose-effect curve for the exposures given at 10 R/min gives a
perfect fit to a curve with a slope of 1.0. ;
Because of the saturation obtained at the highest radiation exposures
there appear to be only minor differences between these three series of
curves. : Because of the large sample sizes in the present experiment we
are confident that the difference between the curves obtained with the
1000 R/min and 10 R/min samples are real. But, I think that it is important
to point out, that in the absence of any additional information or method
of analysis, the smaül differences observed between these 3 dose-effect
curves would probably be attributed to sampling error. The conclusions:
from that part of the experiment presented thus far would, undoubtedly,
have been that a l'eduction in dose-rate from a 1000 R/min to 10 R/min,
results in (a) repair of genetic damage leading to nuclear inactivation
but (b) little or no repair of genetic damage leading to the overall
production of recessive lethal mutations. But because of the additional
methods of genetic analysis available with the ad-3 test system we have i.;
been able to detect a striking dose-rate effect on a particular class
of recessive lethal mutations. About 3600 mutants were selected from
this experiment for analysis. In general, about 300 mutants were
selected, at random, from 4 exposures in each of the 3 treatment series!
Several genetic tests (see Refs. 4 and 9) were then made to distinguish
point mutations at the ad-3A and ad-3B loci from multilocus deletions
covering one or both loci simultaneously.
The details of these tests will be presented separately since they
Involve dose-effect curves for point anutations and multilocus deletions
at each locus individually as well as both loci simultaneously. In this
presentation the data have been combined to simplify the presentation :
and discussion of the general problems.
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مستشملخص
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1. The effect of dose-rate on the frequency of recessive lethal mutations
resulting from point mutation in the ad-3 region
When the frequencies of point mutations obtained with each of the
three treatment series are plotted (Figure 6) no significant difference
is found over the major part of the curves. All three sets of data
give a good fit to a curve with a slope of 1.0. Point mutations
increase linearly with dose and no evidence for repair of genetic
damage leading to this class of recessive lethal mutation is apparent.
with reduction in dose-rate. It is also of interest that the saturation
obtained in the overall forward-mutation curves is apparent in this
particular class of recessive lethal mutation only in the 40 KR exposures
at 1000 R/min.
The effect of dose-rate on the frequency of recessive lethal mutations ;
resulting from chromosome deletion in the ad-3 region
When the frequencies of chromosome deletions in the ad-3 region
obtained with each of the three treatment series are plotted (Figure 7)
no significant difference if found between the two 1000 R/min treatments.
The initial part of both curves gives a good fit to a curve with a slope
.. of 2.0., as expected, but since saturation was found with the 20 and
... 40. KR exposures we get an even more pronounced departure from expectation
with this class of mutations than with the point mutations.
. Much lower forward-mutation frequencies were found with the 10 R/min
exposures than with the 1000 R/min exposures. The dose-effect curve is
complex and appears to be a composite of a l-hit and a 2-hit curve. If :
this is true, then the 5 KR sample should be predominantly l-hit deletions
whereas the 40 KR sample should be predominantly 2-hit deletions. One
might expect a size distribution difference that would be reflected, in:.
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14
turn, in a difference in the relative frequency of different genotypes.
Multilocus deletions covering the ad-34 locus (which is believed to be :
the smaller of the two) are 3 times as frequent in the 5 KR sampie as
in the 40 KR sample. The ratios of the three different genotypes - :
ad-3AR: ad-3BR: (ad-3A ad-3B) IR 181:1:1 in the 5 kr sample and 1:2:6
in the 40 KR sample, thus supporting the hypothesis that the 5KR sample
is predominantly l-hit deletions.
Multilocus deletion mutations show a pronounced dose-rate effect.
The decrease in the frequency of this particular class of recessive
. lethal mutation with a dose-rate of 10 R/min can be attributed to repair
of the single-hit lesions before they can cooperate in pairs to produce
multilocus deletions. In general there is about a 10-fold difference
between the forward-mutation frequencies of the two-hit component of the
10 R/min and 1000 R/min curves.
Decrease of the dose rate by a factor of 100 permits repair of
single-hit events that produce recessive lethal mutations by multilocus
deletion but does not permit repair of those single-hit events that
produce point mutations. With a reduction in the rate of exposure from
1000 R to 10 R/min the forward-mutation rate for the single-hit events.
that cooperate in pairs to produce mutation by multilocus deletion drops
from 9.22 to 2.94 x 10-7 mutants/survivor/R - a 3.2 fold reduction.
The fact that the lesions which give rise to mutation by chromosome
breakage and multilocus deletion are repaired when we lower the dose rate-
whereas those that give rise to point mutation are not, indicates that
they are qualitatively different. Expressed in a different way we feel
that these data provide convincing: evidence that the X-ray induced
lesions that result in chromosome breakage are qualitatively different
from the lesions that result in point mutations.
15
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In this connection, it also seems logical to conclude that the
lesions that result in inactivation are qualitatively similar to those
1
that produce mutation by multilocus deletion. This correlation:
implicates chromosome damage as the dominant lethal event resulting
in the inactivation of heterokaryotic conidia. The reduction in dose-
rate has resulted in similar dose-reduction factors for both effects.
A dose-reduction factor of about 3.2 was found for the single events
resulting in multilocus deletion (Figure 7) and a dose-reduction factor
of about 2.5 was found for inactivation of heterokaryotic conidia (Figure 4).
VII.
The dominant lethal event resulting in inactivation of heterokaryotic
conidia
inacionalina
In experiments with ionizing radiaticns the dominant lethality that
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from the production of a single-hit chromosome aberration. By elimination,
the single-hit event is most probably the production of single-hit terminal
deletions. Since the fusion of the broken ends in such aberrations in
subsequent divisions leads to a breakage-fusion-bridge cyclezo which
slows down cell division, such aberrations are, ultimately, cell lethal.
Also supporting this conclusion is the evidence that ad-3 mutations
never result from terminal deletion. The ad-3A and ad-3B loci 'are..
sufficiently far from the centromere on"linkage Group I that the
production of terminal deletions might be expecied to be the primary
source of ad-3 mutations in a heterokazyon. The same events which, in
combination, produce mutation by multilocus delection, should much more
often produce termined. deletions individually. There seems little doubt
that terminal delations are produced and the fact that such aberrations
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behave as dominant lethals provides an explanation for our failure
to recover ad-3 mutations due to this class of chromosome aberration.
Repair of the single-hit damage which is lethal at ice-water
temperature, leads to slightly higher levels of survival when irradiation
is performed at room temperature and much higher levels of survival. :
when the dose-rate is also reduced 100-fold. In this particular instance,
it appears that the "shoulders" on the survival curves obtained at room
temperature result from repair and do not reflect a change in the number
of targets inactivated. Our interpretation of the change in shape and
slopes of these curves is thus identical with that of Haynes for certain ...
of the survival curves that he obtained after ultraviolet treatment of
' B/r.
A simple way to look at the survival curves of the heterokaryotic
conidia 18 to assume that they have two different components. The number
of survivors observed includes both (1) the total number that are not
inactivated initially by those lesions that result in the production of
single-hit terminal deletions and (2) the total number that now result ::
from repair of this single-hit damage. The relative frequencies of our
estimates of these two different components in each of the survival
curves obtained with the 1000 R/min or 10 R/min exposures is shown in
Figure 8. On this interpretation the "shoulders" on the dose-effect
curves are due to repair at room temperature of damage which is not
repaired at ice-water temperature (compare Figw.es 4 and 8). It is also
evident that the broader "shoulder" on the survival curve obtained with
the 10 R/min exposures reflects much more extensive repair. Our estimates
of the amount of repair are most certainly underestimates. Repair will
not only lead to the elimination of single-hit lesions and higher levels
.
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17.
---
of survival but also to interaction between single-hit lesions to form
two-bit chromosome aberrations (many of which would be expected to behave
as dominant lethals) and lower levels of survival. Finally, our data
show that with an exposure of 40 kR at 10 R/min 91% of the survivors
result from repair of damage that would be lethal with the same exposure
at 1000 R/min at ice-water temperature.
TI
VIII.
The nature of the lesions resulting in recessive lethal mutations
ALI lines of evidence support the conclusion that the lesions that:
give rise to point mutations are qualitatively different from the lesions
that give rise to mutation by multilocus deletion. There is little
doubt that the target involved in the production of point mutations is DNA.
But is this necessarily true for the production of mutations by multilocus
deletion? The most conservative approach is to assume that both types of
mutation result from damage to DNA, but that this damage to DNA is of
two different types. Under the conditions of the present exxperiment
one type of danage to DNA is repaired whereas the other is not.
A partial answer to this problem will undoubtedly result from our
attempts to identify the genetic alterations in those recessive lethal
mutations resulting from point mutations. What is the spectrum of genetic
alterations in X-ray-Induced point mutations at the molecular level? If,
for example, all X-ray-induced point mutations result from minor damage
that alters the pairing properties of the purine or pyrimidine bases,
whereas the mutations resulting from multilocus deletion result from
double-strand breaks, we would have a situation where the initial lesions
are qualitatively different and where they would undoubtedly be subject
.
.
.
-
.
18
!
I
to entirely different mechanisms of repair.
..
IX. Identification of the genetic alterations in point mutations at the
molecular level.
Point 'mutations at the ad-38 locus show allelic complementation,
and specify a linear complementation map. +- Our more recent studies on
nitrous acid-induced ad-3B mutants have shown a definite correlation
between compleanentation pattern and genetic alteration at the molecular
level.+3 Thus, differences in the types of complementation patterns
should reflect a difference at the molecular level in the spectrum of
genetic alterations resulting in point mutation. Experiments are in
progress to study allelic complementation among the point mutations at :
the ad-3B locus resulting from both the 1000 R/min and 10 R/min exposures.
These tests are relatively simple to perform, but any data indicating a
difference in the relative frequencies of each type of complementation
pattern would be, at best, only presumptive evidence of a difference in
: the spectrum of alterations at the molecular level. .
Experiments are also being performed to obtain direct evidence on
the spectrum of genetic alterations resulting in point mutation by means
of tests for specific revertibility. It is yossible to identify the
genetic alteration in individual ad-3B mutants by determining whether they
revert to wild-type after treatment with specific chemical mutagens.
This type of analysis has been completed%924 on 65 ad-38 mutants induced
with 1000 R/min exposures but no data are as yet available on mutants
Induced with 10 R/min exposures. When these experiments have been
completed, it should be possible to determine both whether there is any
repair of any of the damage that results in point mutation and, in particular,
.
:
.
.
-.
**
.
.
whether this repair affects a type of damage that could lead to
chromosome breakage and the production of recessive lethal mutation by :
multilocus deletion.'
X Conclusions
The present experiments have provided convincing evidence for täe.
presence of a VALDEX of repair mechanisms in Neurospora operating at :
either the nuclear or cytoplasmic level. Certainly we have only begun
to analyze the various effects that we have encountered. But nevertheless, ,
we feel that the most important conclusion from the present experiments
is that the X-ray-induced lesions that produce point mutation at specific
loci are qualitatively different from the lesions that produce chromosome
:· breakage.
XI Acknowledgments.
Research jointly sponsored by the National Aeronautics and Space
Administration (NASA Order Number R-104, Task 8), and the U.S. Atomic
Energy Commission wder contract with the Union Carbide Corporation.
XII Summary
A comparison has been made of the effect of reduction in the rate of
X-ray exposures from 2000 to 10 R/min at room temperature on inactivation
of heterokaryotic conidia and the induction of recessive lethal mutations
at specific loci with a heterokaryon of Neurospora crassa. The principal
results are as follows:
(1) Higher levels of survival of the heterokaryotic conidia were
found with 10 R/min exposures than with 1000 R/nin exposures. The dose-
reduction factor is about 2.5
emalisty
r
har
tti-
!
"
-
.
(2) A significant difference was found between the overall induction
curves for recessive. lethal mutation obtained with 1000 and 10 R/min."
Lower frequencies were found with 10 R/min and the slope of the dose.
effect curve was not significantly different from 1.0. The 1:6-fold
· difference in forward-mutation frequency was lower than expected due to
an unexpected saturation of the dose-effect curves with the 20 and
40 KR exposures at 1000 R/min at room temperature.
(3) Genetic analysis of the recessive lethali mutations resulting.
-
-
.
from mutation at the ad-3A and ad-3B loci indicate that (a) there is no
..
dose-rate effect on the induction of that class of recessive lethal
mutations resulting from point mutation and (b) that there' is a 10-fold
difference in the frequency of that class of recessive lethal mutations
resulting from multilocus deletions.-
(4) The experiment demonstrates that the rate of repair of damage
leading to point mutation was markedly different from the rate of repair
of damage causing both multilocus deletions and inactivation of
heterokaryotic conidia.
(5) The finding that the lesions resulting in nuclear inactivation
are repaired to the same extent as the lesions resulting in multilocus
deletion implicates chromosomal damage as the dominant lethal event
responsible for nuclear inactivation in heterokaryotic conidia. The
survival curve data obtained with 1000 R/min and 10 R/min exposures can
be explained by assuming (a) that inactivation results from the production
of single-hit terminal deletions, and (b) that the "shoulders" on the
survival curves result from more efficient repair of this type of genetic
damage at room temperature than at ice-water temperature.
.
:
...21
.
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.
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.
.
XIII References
1. Witkin, E. M., Brookhaven Symposia in Biology 20 (in Press).
2. Setlow, R. B. Brookhaven Symposia in Biology 20 (in Press).
.3. de Serres, F. J. and R. S. Osterbind Genetics 47: 793-796 (1962). -
::.4. Webber, R. B. and F. J. de Serres Proc. Natl. Acad. Sci. U. S.
53: 430-437 (1965). . ;
15. Malling, H. V. and F. J. de Serres Radiation Res. (in Press).
6. de Serres, F. J. and H. G. Kølmark Nature 182: 1249-50 (1958).
7. Brockman, H. E. and F. J. de Serres Genetics 48: 597-604 (1963).
8. Haynes, R. H. . Radiation Res. (Suppl. 6) 1-29 (1966). ..
i 9. de Serres, F. J. Genetics (in Press). .
10. McClintock, B. Genetics 26: 234-282 (1941).
. 11. de Serres, F. J. Genetics 48: 351-360 (1963).
12. de Serres, F. J., H. E. Brockman, W. E. Barnett and H. G. Kølmark
Mutation Research 4: 415-424 (1967).
13. Mailing, H. V. and F. J. de Serres Mutation Research 4: 425–440 (1967).
:14. Malling, H. V. and F. J. de Serres, unpublished observations.
.
$
.
.
.
.
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Table 1
Genetic Composition of Each component of the Dikazyon Used
in Forward-Mutation Experiments
iorito
.
.
,
Linkage Group
IR
III.
Genotype of Bach Component_
Component
I
Component II
(Strain Number 74-0R60-29A) " (Strain Number 74-0R32-16A)
A hist-2 ad-3A ad-3B pic-2
A al-2
ad-
2 ::
.
.
.
-
inos
"
.: pan-2
:
"
Genetic markers are as follows: 1 - mating type; hist-2 - histidine-requiring;
ad-3A, ad-3B, ad-2: adenine-requiring; aic-2 - niacin-requiring; al-2 -
aldino mycelium and conidia; cot - temperature-sensitive morphological (slower
szorta the higher the temperature): inos • Inositol-xequiring; pan
pantothenate-roguarang
•••••••
;-
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Figure Legends
1
I
...c's
Me Swit
Bineider
.
Figure 1 - Survival of heterokaryotic conidia of a dikaryon of Neurospora
crassa on minimal medium after X ray exposure (= Expt. 19-5,
Q = Expt. 12-6, A = Expt: 12-7, A = Expt. 12-10,
: 0 = weighted average (modified from Webber and de Serres 1965).
Figure 2 - Forward-mutation frequencies in the ad-3 region in heterokaryotic
conidia of a dikaryon of Neurospora crassa after X-ray exposure .
(6 = Expt. 12-5, O = Expt. 12-6,' A = Expt. 12-7,. : A = Expt. 12-10,
0 = weighted average) (modified from Webber and de Serres, 1965).
.: Figure. 3 - Forward-mutation frequencies of point mutations (ad-3) and
multilocus deletions (ad-3IR) in the ad-3 region in heterokaryotic ,
conidia of a dikaryon of Neurospora crassa (6 = ad-3mutations,
A = ad-3IR mutations) (modified from Webber and de Serres. 1965).
Figure 4 - Dose-effect curves for inactivation of heterokaryotic conidia
of a dikaryon of Neurospora crassa with dose rates of 1000 R/min'and
10 r/min ( A = 10 R/min, 27°C, 0 = 1000 R/min + 66 hrs posta
irradiation storage, 27°C, C = 1000 R/min # no post-irradiation
storage, 27°C; (6 = 1900 R/min + no post-irradiation: storage, 4°C; *
from Webber and de Serres 1965).
Figure 5 -Frequency of recessive lethal mutations resulting from genetic
alterations in the ad-3 region in heterokaryotic conidia of a
dikaryon of Neurospora crassa at 1000 R/min and 10 R/min
(dotted line = theoretical curve (y = ax + bx?], 0 = 1000 R/min + 66 hrs
post-irradiation storage, D = 1000 R/min + no post-irradiation
storage, A = 10 R/min).
-
-
------ eben
.
ser
H
reconn.
.
-
.
S
+
co
11.
:
Figure 6 - Frequency of recessive lethal mutations resulting from point
'mutation (ad-3) in tre ad-3 region in heterokaryotic conidia of a :
dikaryon of Neurospora crassa at 1000 R/min and 10 R/min''.
(0 = 1000 R/min + 66 hrs. post-irradiation storage,
o = 1000 R/min + no post-irradiation storage, A = 10 R/min;"
dotted line = theoretical curve with a slope of 1.0).
Figure 7 - Frequency of recessive lethal mutations resulting from multilocus
deletion (ad-3-6) in the ad-3. region of Neurospora crassa at 1000 R/min
and 10 r/min (0 = 1000 R/min +966 hrs. post-irradiation storage,
0 = 1000 R/min + no post-irradiation storage, A = 10 R/min;
dotted line = theoretical curve with a slope of 2.0). ..
Figure 8 - Hypothetical single-event and two-event components of the
survival curves for heterokaryotic conidia as affected by dose rate
and irradiation temperature (0 = 1000 R/min +.66 hrs post-irradiation
storage, 27°C, O = 1000 R/min + no post-irradiation storage, 27°C,
: A = 10 R/min, 27°C; solid line = 1000 R/min + no post-irradiation
storage 4°c). .
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DATE FILMED
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