Copyright 0 1990 by the Genetics Society of America
Induced Rates of Mitotic Crossing Over and Possible Mitotic Gene
Conversion per Wing Anlage Cell inDrosophila melanogaster by
X Rays and Fission Neutrons
Toshikazu Ayaki,* Kazuo Fujikawa? Haruko Ryo,* Tetsuo Itoh5 and Sohei Kondol
*Department of Genetics, Nagasaki University School of Medicine, Nagasaki 852, Japan, ?DrugSafety Evaluation Laboratories,
Takeda Chemical Industries, Ltd., Osaka 532, Japan, $Department of Radiation Biology, Faculty of Medicine, Osaka University,
Osaka 530, Japan, and SAtomic Energy Research Institute, Kinki University, Higashiosaka 577, Japan
Manuscript received December 20, 1989
Accepted for publication May 25, 1990
ABSTRACT
As a model for chromosome aberrations, radiation-induced mitotic recombination of mwh andJr
genes in Drosophila melanogaster strain (mwh +/+Jr) was quantitatively studied. Fission neutrons were
five to six times more effective than X rays per unit dose in producing either crossover-mwh/flr twins
and mwh singles-or flr singles, indicating that common processes are involved in the production of
crossover and flr singles. The X-ray-induced rate/wing anlage cell/Gy for flr singles was 1 X
whereas that of crossover was 2 X
theformerandthelatterrate
are of the same order of
magnitude as those of gene conversion and crossover in yeast, respectively. Thus, we conclude that
proximal-marker “flr” singles induced in the transheterozygote are gene convertants. Using the model
based on yeast that recombination events result from repair of double-strand breaks or gaps, we
propose that mitotic recombination in the fly is a secondary result of recombinational DNA repair.
Evidence for recombinational misrepair in the fly is given. The relative ratio of radiation-induced
mitotic crossover to spontaneous meiotic crossover is one order of magnitude higher in the fly than
in yeast and humans.
C
HROMOSOME aberrations are a well-documented cause of human genetic disease (GARDNER 1983;SCHINZEL1984). Since the early days of
classical genetics, the study of chromosome aberrations in higher organisms has been a major branch of
modern biology. However, the molecular mechanisms
of chromosome aberrations are still not understood
(SAVAGE 1989). A major obstacle in the progress of
molecular biology of chromosomeaberrations
in
higher eukaryotes is the lack of appropriate mutant
genes that interfere with production of chromosome
aberrations.
In
Saccharomyces
cerevisiae,
however,
thereare many mutantgenesthatinterfere
with
meiotic and mitotic recombination (reviewed in
HAYNESand KUNZ 1981 ; KUNZ and HAYNES 1981).
Recent studies using such mutants have led to the
hypothesis that meiotic or mitotic recombination results from repair of double strand breaks or gaps in
DNA(HASTINGS1988;ORR-WEAVERand SZOSTAK
and STAHL1988). This hypothesis is
1985; THALER
consistent with the observations that mitotic crossing
over and mitotic gene conversion in yeast (NAKAI
and
MORTIMER 1967; RAJU et al. 197 1 ; UNRAU1986,
1987) and chromosome aberrations in higher organisms (BENDER
1970; GOODHEAD
1982; LEENTHOUTS
and CHADWICK 1978; SASAKI
et al. 1989) are induced
more efficiently by densely ionizing radiation (fast
neutrons, heavy charged particles, ultrasoft X rays)
Genetic?,126: 157-166 (September, 1990)
than sparsely ionizing radiation (X or y rays). This is
because, at an equal absorbed dose, densely ionizing
radiationproducesdouble-strandbreaksmore
efficiently and in a more clustered fashion than sparsely
ionizing radiation (FRANKENBERG
et al. 1986; GOODHEAD 1982; HOLT1988; LEENTHOUTS
and CHADWICK
1978; SASAKIetal. 1989). However, mitotic recombination events in yeast may differ in some respects
fromthose in humans due to monocellular versus
multicellular organization and because of the difference of two orders of magnitude in both genomic
DNA content and meiotic recombination frequency
(CATCHESIDE
1977).
Drosophila melanogaster is a good animal model, as
our knowledge of its genetic constitution is extensive,
its DNA content is intermediate between yeast and
humans, and yet its chromosomes (except the fourth)
are similar in structure and of the same order of
magnitude in size as human chromosomes (reviewed
in ALBERTS
et al. 1989).
Mitotic recombination was first discovered in D.
melanogaster by STERN( 1 936). X-ray-induced mitotic
crossing over has been extensively used by GARCIAand his associates (1971a,b, 1972, 1974)and
BELLIDO
others (fora review see POSTLETHWAIT
1978) to study
the development of various imaginal discs in the fly.
Using a transheterozygous strain with the same genotype as that used by GARCIA-BELLIDO
and DAPENA
T. Ayaki et al.
158
(1 974),we measured the rate
of crossing over induced
X rays or fission neutrons.If
induced rates/anlage cell/Gy of crossover in the fly
are quantitatively close to those of crossover in yeast
(NAKAIand MORTIMER1969; RAJUet al. 1971), such
agreement suggests that our knowledge of the mechanism of mitotic recombination in yeastmay be applicable to thatin the fly.
In yeast, more data a r e available on gene conversion
than on crossing over. In the fly, however, no clear
cutevidence is available foroccurrenceofmitotic
gene conversion except for suggestiveevidence reported by STERN(1969) and TOKUNACA
(1973), although occurrence of meiotic gene conversion is well
1984; CHOVNICK, BALLANdocumented (CARPENTER
TYNE and HOLM197 1; CURTIS et a/. 1989; HILLIKER
and CHOVNICK1981). GARCIA-BELLIDO
(1972) reported that upon X-irradiation of flies heterozygous
for multiple recessive markers in trans or cis constitutions, proximal marker singleswere induced several
hundred times more frequently than theoretical estimates basedon double crossing over.If such proximal
marker singles primarily represent gene convertants,
be of the
their induced rates/anlage cell/Gy could
same order of magnitude as the reported values for
induction rate per Gy of mitotic gene convertants by
X-irradiation in yeast (NAKAIand MORTIMER 1969).
This idea was tested in this study.
by irradiationwith
MATERIALS AND METHODS
Stocks and somatic cell markers used: D. melanogaster
strains with the genotypes of y;mwhju (GRAFet al. 1984) and
jZr3/TM3, Ser (GRAFe l al. 1989) were obtained from F. E.
WURCLER,Zurich University. The third chromosome from
the former stock and a spaPo‘-bearingfourth chromosome
from a laboratory stock were introduced into a Canton-S
wild type strain to construct stock mwh ju;JpaP”’. Crossing
this stock with stockflr3/TM3, Ser, we obtained FI progeny
with heterozygous genotypes mwh julflr3 and mwh julTM3,
Ser. Hereafter these are referred to as mwh +/+ f l r and
mwhlTM3, respectively. In the adultwing, flies homozygous
for the recessive marker mwh produce three or more hairs
per cell, whereas a single, smooth hair per cell is produced
in normal flies. Homozygosity for marker f l r is lethal; flies
heterozygous for markerflr areviable. If homozygous( j l r /
J r ) cells are somatically produced in heterozygous flies, they
are viable as long as the clone sizeis small, and produce
misshapen “flaring” hairs in the adult wing. Loci mwh and
J r are located in the left arm of third chromosome at map
positions 0.0 and 39.0, respectively. Locus mwh is located
cytologically in segment 61E-62A of the salivary chromosome map (LEWIS1969). Chromosome TM3is a multiply
inverted third chromosome. Among breakpoints between
the centromere andthe mwh locus inchromosome TM3, the
most distal one from the centromere is located at segment
65E; it is the nearest to the mwh locus.
For the description of the mutants and the balancer used
exceptflr’ (GARCIA-BELLIDO
and DAPENA 1974),
see LINDSLEY and GRELL
(1 968).
Culture of larvae: Emerged females from stock mwh
jv;spaP”‘ and males from stock flr3/TM3, Ser were kept
separately as virgins for 3-4 days at 25”. They were then
mated at a ratio of 40 females to 40 males in 28 mm x 110
mm polycarbonate culture vials containing 20 ml Drosophila
medium (powdered yeast “Ebios”60 g, glucose 60 g, agar 8
g, and propionic acid 5 ml per 1000 ml water) for 24 hr,
and then transferred to fresh culture vials to oviposit for 4
hr. After oviposition, parental flies were discarded and eggs
were allowed to develop. Larvae were sampled from culture
vials on day 4 after oviposition. They were transferred to
plastic petri dishes (60 mm X 13 mm) containing 5 ml
medium. The cover of each dish had a hole covered with a
piece of nylon gauze to prevent anoxia when larvae placed
in these dishes were piled upon each other and kept for
several hours in a narrow holeof a nuclear reactor for
irradiation. Similarly prepared larval sampleswere used for
X-irradiation.
Irradiation: The Toshiba X-ray generator used was operated at 180 kVp and 20 mA with 1 mm AI filter; the dose
rate used was 0.49 Gy/min as measured with a Victoreen
cavity chamber. A quarter of a total dose was given four
times at intervals of 80 min; the first and thelast fractionated
irradiation were approximately synchronized to the start
and the end of irradiation with neutron-? mixed radiation
inside a nuclear reactor.
The reactor used is called the University Teaching and
Research Reactor, type B, and was manufactured in 1958
by Advanced Technology Laboratories (division of American-Standard), Mountain View, California. It operates at a
maximum power of 10 watts witha pair of tanks containing
‘J99
--.U-enriched uranium fuels immersed in a small quantity
of water. Fission neutrons generated from 2‘’5U fuels are
slowed down by the water and then reflected by graphite
piles. Because the twin fuel tanks are located 46 cm apart
from each other, a fairly large dry area is available for
irradiation of biological samples placed at the center of tLe
reactor with an approximately uniform dose-rate.
The reactor was operated at 1watt for irradiation of flies.
Dosimetry offast neutrons against contaminated gamma
rays was done using a pair of ionization chambers (Far West
Tech., Goleta, California) (HOSHIet al. 1988). To each dish
containing larval samples, we attached plastic neutron detectors “TS16N” (Nagase-Landauer, Ltd., Tokyo) to monitor in situ neutron dose and thermoluminescent rods “UD170L” (Matsushita Electric Ind. Ltd., Osaka) to monitor in
situ gamma dose. Dose rates averaged over 10- 12 dishes
containing larval samples were 0.43 (tissue-Gy/hr) for total
neutron-gamma mixed radiation and 45% of 0.43 Gy/hr
was due to fission neutrons (YASUBUCHIet al. 1989). Graded
dosesweregiven to samples by changing the irradiation
period.
Immediately aftertheend
of irradiation, larvae were
transferred to vials containing fresh medium and cultured
at 25’ until adults emerged.
Larval age at irradiation: Irradiation of larvae was
started at age 96 f 2 hr post-oviposition. This late third
instar stage was chosen for irradiation because larvae Xirradiated at this stage yielded the highest frequency of
mwh/flr twins, which was as high as that for mwh singles
(data not shown). We judged that this was an optimal stage
for quantitative analyses of radiation-induced yields of mwh
and flr spots on the basis of the following results that were
obtained after X-irradiation with 9.5 Cy of larvae at different stages. Large mwh spots showed an approximately exponential increase in their induced frequency (spots/wing)
with increase in the time at irradiation from immediately
after hatching to immediately before pupation. In contrast,
mwh/flr twinsshowed a specificstage dependence; the
frequency showed no increase above control level after
159
Mitotic Recombination in Drosophila
irradiation at 26 and 50 hr postoviposition,barely rose
above control level after irradiation at 74 hr, and thereafter
increased sharply, attaining a maximum value at 92-98 hr.
The frequency of flr singles alsorose above the spontaneous
level only when X rays were given at late stages. The mean
clone size oflarge mwh singles and thatof twin spots induced
by X-irradiation decreased exponentially with increasing
larval age at X-irradiation whereas the mean clone size of
flr singles was less dependent on the larval age at X-irradiation.
Observation of mutant spots:Somatic recombinant spots
on the adult wing were scored following the method of
GRAFet al. (1984). Flies that emerged were preserved in
70% ethanol until use. The preserved wings were washed
with distilled water and then mounted on glassslidesin
Fauer's solution (gum arabic 30 g, glycerol 20 ml, chloral
hydrate 50 g, water 285 ml) to score mosaic spots on each
wing under a microscope at 400X magnification. Only femaleswere used; they were composed oftwo genotypes,
(mwh +/+Jr) and (mwhlTM3). On the (mwh +/+Jr) wing,
three types of mosaic spots appear: (i) mwh/flr twin spots
composed of mwh spots and flr spots appearing at an adjacent area; (ii) mwh single spots without accompanying flr
spots in an adjacent area; and (iii) flr single spots without
accompanying mwh spots in an adjacent area. We adopted
the conventional classification (GRAFet al. 1984) ofmwh
singles into large ones made ofthree ormore cells and small
ones made of one or two cells.This classificationis not trivial
as small mwh singles comprise the majority of spontaneous
mwh singles. Smallflr singles are very rare (GARCIA-BELLIDO
and DAPENA1974; GRAFet al. 1984). Inversion heterozygotes (mwhlTM3) were used to estimate the fraction of mwh
spots generated by recombination events as inverted chromosomes are known to suppress the occurring of mitotic
crossovers (BECKER1976; MERRIAMand GARCIA-BELLIDO
1972).
Estimation of mitotic recombination events per wing
anlage cell: T o perform a quantitative comparison of mitotic recombination events between D. melanogaster and S.
cereuisiae, the frequencies, F , of recombinant spots per wing
observed in flies must be converted to the frequencies,f, of
mitotic events per cell in the imaginal disc.
Number n of anlage cells in the wing imaginal disc increases exponentially with larvalage t (GARCIA-BELLIDO
and
MERRIAM197 la,b; POSTLETHWAIT
1978):
n=
2"l
(1)
where u is rate of cell divisionper unit time. We assume that
irradiation of the disc with dose D Cy produces a recombinant (mwh or flr) cell among n anlage cells at a frequency
of bnD, as follows:
F = F,
+ bnD = F, + b ( n ) D ,
(2)
where F is the frequency of mutant spots per wing for
irradiated flies, F, the frequency of spontaneous mutant
spots per wing, and b represents the induced rate/wing
anlage cell/Gy for production of mwh or flr spots. The third
formula is an expression for actual cases, inwhich total
number n of anlage cells in the wing imaginal disc at the
time of irradiation fluctuates around mean value ( n ) . To
estimate ( n ) , we assume that an ancestral mutant cell produced in the wing disc by radiation expands its clone size at
the same rate u as that of normal anlage cellsgivenin
Equation 1. Then, final size S of an rnwh or an flr clone
generated by radiation is given as follows:
s = 2"("') = N / n
(34
where T is the age of finaldivisionofcellsin
the wing
imaginal discand N (=2"7) is the total number of cells in the
adult wing.In fact, the mean clone size of mutant spots
decreased approximately exponentially with increasing time
t at X-irradiation with an equal dose of 9.5 Cy (see section
"larval age at irradiation"). Actual mutant clones in irradiated flies include spontaneous mutant spots; actual clone
sizes show wide variation. Mean clone size (S) for induced
clones may be given by
,=1
where S,is the subtotal number of mutant cells in mutant
clone i, K the total number of mutant clones detected, z the
number of wings observed-all for irradiated flies-and symbols with subscript o denote parameters of control flies.
Using the (S) value thus calculated, we can estimate mean
value ( n ) as follows:
( n )= N/(S).
(4)
Combining Equation 4 with the third formula of Equation
2, we obtain the following equation for radiation-induced
frequency J; of recombinant-producing events per anlage
cell:
J; = bD = ( F - F , ) ( S ) / N .
(5)
RESULTS ANDDISCUSSION
Frequencies of mitotic crossing over events per
wing anlage cell induced by X rays and neutron-7
mixed radiation: T h e frequencyf; of mitotic recombination events in anlage cells, which were induced by
irradiation at larval stages and eventually led to the
observed mwh/flr twins or large mwh singles in the
adult wing, was estimated using Equation 5. The N
value in Equation 5 , ie., the total number of cells in
the wing blade, was estimatedfrom the following
formula:
N = 2dA
(6)
where d is the wing hair density per 0.01mm2, A
(mm2) the whole area of the wing blade except for
regions A' and E' (GARCIA-BELLIDO
and MERRIAM
1971b) and factor 2 is introduced to account for two
layers of the wing, dorsal and ventral. The d values
were estimated from microscopically observed numbers of hairs per 0.0226 mm2 at ten dorsal sites in
regions A, C, C', D, D' and E (GARCIA-BELLIDO
and
MERRIAM1971b). The observed A and d values of
flies irradiated with X rays or neutron-? mixed radiation were not significantly differentfrom those of
control flies. N values thusobtained were 2 x lo4
(=( 1.99 f 0.15) X 104]for the mwh + / + j r strain and
1.8 X lo4 (= (1.78 k 0.08) X lo4] for the mwhlTM3
strain.
Table 1 summarizes the J; values forlarge mwh
singles, mwh/flr twins and smallmwh singles estimated from observed frequencies F of mutant spots
per wing. It also givesJ values for flr singles similarly
T. Ayaki et al.
160
TABLE 1
Conversion of observed frequenciesF of mutant spots per wing to induced frequencies4 of recombination events per cell
__
spots
T \ p C ~1iInIII;lIlI
__
Frequency of mutant
spots per
wing,
F, or
F
Subtotal of mutant
cells, CS:
G ) b
0.04
0.9 1**
1.67**
1.12**
1.go**
116
250 1
3042
3868
4456
7.9
7.6
7.6
7.9
3.4
6.2
4.1
7.3
0
0.12
2 (X)
4 (X)
0.84 (n + y)
1.66 (11 + y)
1.29**
2.38**
1.91 **
2.95**
424
3344
4321
7167
6966
7.3
7.5
8.3
7.9
4.3
8.5
7.4
11.2
858
1324
945
1625
1416
1.5
1.7
1.5
1.5
0.9
+ y)
+ y)
1.54
2.68**
2.69**
2.56**
3.33**
+ y)
+ y)
0.005
0.11**
0.16**
0.14**
0.22**
11
268
239
435
378
6.0
7.2
5.9
Kadiation dose
(CY)
0
2 (X)
4 (X)
0.84 (n
1.66 (n
+ y)
+ y)
0
2 (X)
4 (X)
0.84 (11
1 .66(11
0
2 (X)
4 (X)
0.84 (11
1.66 (n
Average
size of
induced
clones,
__
Induced
frequency of
recombination per
lo4 cells,f;'
1 .o
0.8
1.3
6.6
0.4
0.5
0.5
0.6
Number o f wings scored for control (2.) was 425 and those for irradiated groups( 2 ) were 351 for 2 Gy (X) and 241 for 4 Gy (X), 454 for
0.84 Gy (11 y) and 298 for 1.66 Gy (11 y) where (X) is X rays and (n y) is neutron-y mixed radiation.
" The CS,values for mwh/flr twins are those for mwh partners; the values for flr partners were 82-86% of those for mwh partners.
' Calculated from observed values CS,,z, z,, k , k. using Eq. (3b) in text.
Calcul;1ted frolu F ,F(,and ( S ) and N values, using Equation 5 in text.
** Significantly higher tlxm control frequency F, at P < 0.01 by x2 test.
+
+
estimated from observed flr singles per wing.
The obtained J values for twin spots are plotted
against the dose of X rays and neutron-gamma mixed
radiation in Figure1 ; they increased linearly with
increasing dose of either X rays or neutron-y mixed
radiation.
It was previously established by GARCIA-BELLIDO
and DAPENA(1974) that mwh/flr twins in the (mwh
+/+JZr) strain result from mitotic crossover.
The conclusion that large mwh singles composed of
three or more mwh cells mostly result from crossing
over is supported by the following results. X-rayinduced rates, b, (/lo4 cells/Gy), for large mwh spots
(total of mwhfflr twins and large mwh singles) in (mwh
+/+ j l r ) , m w h l T M 3 , and mwh/TMI strains are 3.7,
0.42 and 0.25 (Table 2), in the ratio of 1:0.11:0.07.
On the other hand, the ratios of the left arm of the
normal third chromosome to the normal part retained
in the left arm of chromosome T M 3 and to that of
chromosome TMI are 1:0.17:0.07 as estimated from
the mitotic chromosomemap (BECKER1976).It is
known that somatic recombinationeventsafter
Xirradiation occur uniformly along the entire lengthof
normalchromosomes
(BECKER1976; GARCIA-BELLIDO 1972).
+
81
92257
DOSE ( G y )
FIGURE1.-Frequencies
per wing anlage cell of mitotic crossover,f, to produce mwh/flr twins in the rnwh + / + J r wing plotted
against the dose of Xrays (0)and neutron-y mixed radiation (e).
Symbol 0 stands for spontaneous frequencies. Thefvalues indicated
at 10 points (0,0 , @) are calculated from the values of twins per
wing observed in two sets of experiments following the method
indicate
shown in Table 1 . Numerical figures attached to each point
subtotal numbers of mwh partner cells of twins observed at the
indicated doses of radiation. Curve A is theoretical dose response
to fission neutrons constructed using experimental frequencies for
neutron-y mixed radiation (e)after correction for contribution by
contdlllinated y rays, i e . , using the RBE value for twins in Table 2.
Mitotic Recombination in Drosophila
161
TABLE 2
Relative biologicaleffectiveness (RBE) of fission neutrons for productionof mwh/flr twins, large mwh singles and flr singles
~~
~~~~~~~~~~~~~~
Induced rate" (IO-4/cell/Gy) by
Type of induced somatic mutations
D. melanogaster
strain
mwh/flr twins
Large mwh singles
flr singles
Large mwh singles
Large mwh singles
mwh + / + j l r
mwh + / + f l y
mwh + / + f l y
mwhlTM3
mwh/TMI'
Neutron-y
mixed
radiation, b.,
4.63 f 0.20
7.88 f 0.55
0.44 f 0.05
1.14 f 0.04
NDd
X rays, b,
1.61 f 0.05
2.12 f 0.08
0.13 f 0.02
0.42 f 0.03
0.25 f 0.02
Estimate for induced
rateb (10-4/cell/Gy)
by '"U fission neutrons, b.
RBE, b./b,
8.18 k 0.43
14.66 & 1.03
0.83 k 0.12
2.02 k 0.32
5.1 f 0.3
6.9 f 0.6
6.2 f 1.1
4.8 f 0.8
ND
ND
Coefficients of linear regression fitted by the least square method to the induced frequencies,$,given in Figure 1 and Table 1.
Calculated from b,, and b, values assuming that X and y rays have an equal b value and using Equations 7 and 8 in text.
mwh/TMI larvae were obtained from the cross of y; mwh j v females and y; D p ( l : j ) SI?', y + f l r / T M I , Mi. ri sbd' males (GRAFet al. 1984).
T h e parental male strain was obtained from F. E. WURGLER,
Zurich University.
Not determined.
Relative biological effectiveness (RBE) of fission
neutrons for induction of twins, large mwh singles
and flr singles: The$ values given in Figure 1 for
mwh/flr twins, and those for large mwh singles and
flr singles given in Table 1, arefitted to linear regression by the least-square method, using the reciprocal
of the variances of observed frequencies asweighting
factors. Obtained regression coefficients are the values of b (/cell/Gy) defined by Equation 5. The b values
are summarized in Table 2; b,, is induced rate/cell/
Gy by neutron-y mixed radiation and b, induced rate
by X rays.
If fraction r of a totaltissue dose of neutron-y mixed
radiation is contributed by fission neutrons, the b,,
value can be approximated by
b,, = (1
- r)bg + rb,,
(7)
where bg is induced rate by gamma rays. Assuming
that bg equals b,, from Equation 7 we have
bn
= bz
+ (bn+g - b x ) / r -
(8)
T h e ratio b,/b, gives the RBE of fission neutrons.
T h e RBE values of fission neutrons thus obtained for
the experimental r value of 0.45 were 5.1, 6.9, and
6.2 for induction of mwh/flr twins, large mwh singles
and flr singles, respectively, in the inversion-free strain
and 4.8 for induction of large mwh singles in the
inversion-heterozygous strain (Table 2).
Evidence that flr singles are mitoticgene converJr)
tants: Induction of flr singles in the (mwh
strain by X rays cannot be explainedby double crossover events. The X-ray-induced rate (/cell/Gy) of
crossing over between the centromere and locus J r ,
1.6 X
(Table 2), multiplied by that between J r
and mwh loci, 2.1 X
(Table 2), gives 3.4 X lo-';
this product is smaller by a factor of about 400 than
the induced rate 1.3X lo-' for induction of flr singles
(Table 2). The same conclusion was previously made
by GARCIA-BELLIDO
and DAPENA(1974) and they
interpreted this to indicate thatsingle crossover events
+/+
between theJr locus and the centromereoccasionally
generate recombinants that fail to express phenotypically the homozygous marker mwh. We conclude,
however, that flr singles resultfrom mitotic gene
conversion events, on thebasis of the following results:
(1) The RBE value of fission neutrons, 6.2 k 1.1, for
induction of flr singles agrees, within the limits of
experimental error, with those for mwh/flr twins and
large mwh singles, 5.1 k 0.3 and 6.9 k 0.6, respectively (Table 2), an indication of common processes
for production of these three types of mutant spots.
(2) The X-ray-induced rate/cell/Gy for flr singles, 1.3
X
is in therange of X-ray-induced rates0.05-3
X
of mitotic gene conversions at various heteroallelic loci in yeast (MANNEYand MORTIMER 1964;
NAKAIand MORTIMER1967; RAJUet al. 1971; UNRAU
1986). This agreement does not appear to be fortuitous. X-ray-induced rates/cell/Gy for large mwh spots
in the (mwh +/+fir) and (mwh/TMI) strains are 3.7 x
10-4 and 2.5X 10-5 (Table 2), respectively. This range
of variation is close to the range of the highest and
X
and 8 X
(/cell/Gy), for
lowest rates,
1.6
induction of mitotic crossovers in yeast (NAKAI and
MORTIMER1969). (3)In yeast, fast neutrons and other
densely ionizing radiations are two to seven times
more effective for induction of mitotic gene conversions and mitotic crossovers than X and y rays (MORTIMER, BRUSTAD
and CORMACK 1965;NAKAI and
MORTIMER 1967; RAJU et al. 1971;UNRAU1986).
These results are compatible with the RBE value of
five to six of fission neutrons for induction of mitotic
recombination events in the fly (Table 2). (4) Using
strains with various recessive markers (includingdistal
marker y or mwh) in trans and cis constitutions, GARCIA-BELLIDO
(1 972) and
GARCIA-BELLIDO
and DAPENA
(1 974) compared X-ray-induced frequencies of crossing over (mutant twins or multiply mutated clones in
trans or cis heterozygotes) with those of proximalmarker (sn, J J r or j v ) singles; observed ratios of
proximal-marker singles to crossover spots in nine
162
T. Ayaki et al.
experiments varied from 0.022 to 0.12 with an average value of 0.059 f 0.029. Our ratios offlr singles
to crossover spots induced by neutron-y mixed radiation and X rays were 0.045 and 0.04-0.05, respectively (data from Table 1). In the S. cerevisiae strain
with the cis-heterozygous genotype ad5,? ly5 ty3 metl3
acrq tr5 le,/ADT,, LYs T Y j M E T l 3 ACsz TR5 LE, on the
left arm of the seventh chromosome, the frequency
ratios of singles expressing proximal-marker le,, tr5,
acrz, metl3, ty3, and ly5 to crossing over of the most
distal marker ad5,7 after X-irradiation are 0.01, 0.04,
0.02, 0.06, 0.03 and 0.02,
respectively (datafrom
NAKAIand MORTIMER 1969).Thus, six frequency
ratios in yeast and ten frequency ratios in the fly are
quantitatively close to each other. Thisapproximation
strongly suggests that not only flr singles induced in
trans-heterozygotes but also sn, f, flr and j v singles
induced in trans- or cis-heterozygotes are gene convertants.
Comparison of the radiation-induced rateof mitotic crossing over among yeast, flies and humans:
In humans, atomic bomb radiationinduces crossovers
of M/M types at the rate of 1-1.4 X 10-5 (/cell/Gy)
in blood cells of atomic bomb survivors with heterozygous genotype M/N for glycophorin A (KYOIZUMI
et al. 1989a; LANGLOIS
et al. 1987). We quantitatively
compare whether radiation-inducedmitotic crossover
occurs more frequently in humans than in the fly or
yeast. Assume thatheterozygotes
a/+ with distal
marker a at chromosomal distance L from the centromere produce induced rate b (/cell/Gy) of crossover recombinants after X-irradiation. Then, b may
be given by
b = cr,itdL = cd(r,,,it/rmei)M,
(9)
where d is density of X-ray-induced recombinogenic
lesion per unit chromosome length per Gy and rmit
density per unit chromosome length
of mitotic recombination hotspot (EDELMANN
et al. 1989; THOMAS
and
ROTHSTEIN1989; VOELKEL-MEIMAN,
KEIL and ROEDER 1987)toconvertarecombinogenic
lesion to
mitotic crossover and c proportionality constant. If we
use an average value for spontaneous meiotic recombination rate per unit chromosome length, rmei(=reciprocal of theaveragenumber
of base pairs per
centiMorgan),then we can convert L into M , the
distance in units of centiMorgan, obtaining the third
formula of Equation 9. Because d and c may not differ
greatly among different eukaryotic species, the third
formula of Equation 9 implies that b/M in a species
depends on the ratio r,,it/rnmei
in that species. Table 3
compares the radiation-induced rate of mitotic crossover among yeast, fruit flies and humans; the rmit/rmei
value for the fly is larger by one order of magnitude
than those for yeast and humans. This conclusion
reminds us of Muller’s saying in his Nobel prize lecture (MULLER 1946): “Drosophila, an organism in
which the synaptic forces are known tooperate
strongly even in somatic cells, than in other organisms
tested.” This conclusion is consistent with the intimate
somatic pairing of homologous chromosomes during
early mitotic prophase in Drosophila (BECKER1976;
HILLIKER
and APPELS1989; METZ 19 16); the
intimate
pairing may exist during interphase as well, providing
opportunity for a high induction rate
of gene conversion after irradiation.
Mechanism of mitotic recombination in relation
to DNA repair in Drosophila: Mitotic recombination
events result from repair of double-strand breaks or
gaps in DNA. This is the model recently supported
by various results in fungi (HASTINGS1988;ORRWEAVER
and SZOSTAK
1985; RAY,
MACHINand STAHL
1989; THALER
and STAHL1988) and cultured mammalian cells (BRENNER,SMIGOCKIand CAMERINIOTERO 1986). Keeping this model in mind, we next
consider our findings that fission neutrons are five to
six times more effective than X rays in generating
crossovers (mwh/flr twins and large mwh singles) and
gene conversions (flr singles) (Table 2). Double-strand
breaks of chromosomal DNA induced by fission neutronsarenot
only moreabundantbut
also more
densely distributedthan those induced by X rays
(HOLT1988; LEENTHOUTS
and CHADWICK1978).
Hence, the former lesions are less often repaired by
the excision repair and postreplication repair processes that operate during GI and
S phases (FRIEDBERG
1985; LEENTHOUTS
and CHADWICK1978;RUBIN
1988; WITKIN1976). X-ray-induced DNA repair persists until the subsequent cell cycle after irradiation
(PEAK,PEAKand BLAZEK1988).Consideringthese
fragmentary results, we assume that unrepaired double-strand breaks carried over into the G2 phase are
dealt with by recombinationalrepair.
Recombinational repair of X-ray-induced damage was first shown
by NAKAIand MATSUMOTO(1967) using a recombinationless (later named rad51) strain of S. cerevisiae
and the model of recombinational double-strand re(1976). Most, but
pair was first proposed by RESNICK
not all, defective meiotic genes reducemitotic recombinational DNA repair as indicatedby reduced diploidy resistance against the lethal effects of alkylating
agents and radiation (HAYNES and
KUNZ 1981; SAEKI,
MACHIDAand NAKAI 1980). Inyeast, however, available results favor the model in which mitotic recombination occurs more frequently in the GIphase than
in the G2 phase (HASTINGS1988; ORR-WEAVER and
SZOSTAK
1985; WILDENBERG
1970). By contrast, in D.
melanogaster, since the first discovery of mitotic recombination by STERN (1936),its occurrence at the
four-strandstage, i.e., from GP phase to early prophase, has been tacitly assumed (BECKER1976).
KONDO(1989) proposed the hypothesis that large
mwh spots are induced after X-irradiation as a result
Recombination
Mitotic
in Drosophila
163
TABLE 3
Comparison of X-ray-induced rate per centimorgan
of mitotic crossing over among
S. cereuisiae, D. melanogaster, and Homo sapiens
~
~~
Cell marker in
heterozygote
Organism
S. cerevisiae
D. melanogaster
adr.7
acrp
mwh
W
Homo sapiens
M
Distance between
centromere and
cell marker,
M (cM)”
170
58
50
60
70‘
Induced rate of
crossover events
Induced rate
between centromere and the
per centimorgan,
marker,
b ( l c e centimorganb
Wy)
Reference
blM
1.6x
6.7 X
3.7 x
7.2 x
1.4 X 1 0 - 5 ~
9.4 X
1.2 x
7.4 x
1.2 x
2.0 X
10”
IO+
IO+
10”
Base pairs per
6X
6X
7X
7X
1x
IO3
10’
lo5
IO5
IO6
NAKAIand MORTIMER
(1969)
Present study
BECKER
(1976)
KYOIZUMIet al.
(1989a)
O’BRIEN
(1987).
CATCHESIDE
(1977).
‘ Approximate value from KENNETH,KLINCERand RUDDLE1989.
After exposure to Hiroshima atomic bomb radiation.
of Gp-specific recombinational repair of potentially
lethal double-strand breaks, reasoning as follows. Excisionless strains mus201/mus201 and mei-9 produce
gene mutations at higher frequencies than repair proficient strains after treatment with UV and alkylating
and KONDO1986; RYOand KONDO
agents (FUJIKAWA
1986). DNA lesions induced by UV and alkylating
agents are repaired by excision repair in G1 phase and
unrepaired DNA iesions are converted into mutant
DNA sequences during DNA synthesis (FRIEDBERG
1985; WITKIN1976). Thus, we have an axiom that
repair of mutagenic DNA lesions can reduce themutagenicity only when repair occursprior tocompletion
of the mutagenesis process. Strains with mutation mei4 1 and its allele muslO4 are defective in postreplication repair (BOYDand SETLOW1976) and give lower
frequencies of gene mutations than repair-proficient
strains after treatment with X rays, UV and alkylating
agents (FUJIKAWA
and KONDO 1986; RYOand KONDO
1986). These results are compatible with the axiom
because induced gene mutations mostly result from
errors in postreplication repair of mutagenic DNA
lesions (FRIEDBERG
1985; WITKIN1976). In contrast,
large mwh spots are induced at six and twelve times
higher frequencies in strains mei-41 and mei-9 mei-41,
respectively, than in a repair-proficient strain (Yoo,
RYO and KONDO 1985). Applying the axiom to this
result, we deduce that mitotic recombinational repair
of X-ray-induced double-strand breaks, which results
in generation of crossover, i.e., mwh spots, occurs or
completes its final step after termination of post-replication repair, i.e., in GPphase. This hypothesis is
compatible with the intimate somatic pairing of homologous chromosomes during early mitotic prophase
(BECKER1976; HILLIKERand APPELS 1989; METZ
1916).
Recombinational DNA misrepair: If mitotic recombinational repair, a G2-specificone, works to efficiently convert densely distributeddouble-strand
breaks to tolerable DNA aberrations (crossovers, gene
conversions etc.), it must be accompanied by repair
DNA aberrations
errors,producingsemitolerable
such as semilethal chromosomal aberrations at the
cellular level. It has been well established by MERRIAM
and GARCIA-BELLIDO
(1972), HAYNIEand BRYANT
(1977), BAKER,CARPENTER
and RIPOLL(1 978),KENNISON and RIPOLL(198 1) and GRAFet al. (1 984) that
semilethal chromosomal aberrations, such as lossof
the normal third chromosome or deletion of the terminal segment including the normal allele to mwh,
are, atleast partly, responsible for occurrenceof small
mwh clones. Their reasoning is essentially as follows:
mwh anlage cells bearing semilethal chromosomal aberrations will be eliminated by competition with normal (mwh/+) anlage cells for clonal expansion during
larval development, unless they are produced at late
stages. T h e frequency of spontaneous smallmwh
clones of size one ortwo in the inversion-heterozygous
strain mwhlTM3 is 40-50% of that in the inversionfree strain (mwh + / + J r ) (data not shown). This difference indicates that about half of the small mwh
clones in the normal strain may result from errors in
spontaneous recombinational repair. Most small mwh
clones in the normal strain have low viability (BAKER,
and RIPOLL1978).
CARPENTER
If small mwh clones are mostly caused by mitotic
recombination,these will beproducedmoreabundantly after irradiationwith fissionneutrons thanwith
X rays as is the case for production of large mwh
singles. As shown in Table 1, theinduced frequencies
per lo4 anlage cells of small mwh singles by 0.8 Gy of
fission neurons plus y rays and by 2 Gy of X rays are,
respectively, 0.8 and 0.9. Hence, using Equation 8
and assuming a linear dose response, we obtain 3.7
for the RBE value of fission neutrons for inductionof
small mwh clones.
Human diseases associated with mitotic recombination: Evidence thatspontaneous mitotic crossing
T. Ayaki et al.
164
over or gene conversions are involved in the development of cancer cells from cells heterozygous for
recessive oncogenes has been accumulating (ALBERTS
et al. 1989; KNUDSON1986; KOVACS,WILKENSand
POPP1988; SCRABLE
et al. 1987; WESTONet al. 1989).
One of the major characteristicsof Bloom's syndrome
is an increasedspontaneous rate of in vivo mitotic
crossing over (LANGLOIS
et al. 1989; KYOIZUMIet al.
1989b).
Use of an induced rateof crossover to produce M/
M mutant cells and those of somatic deletions from
M/N to M/O or N/O mutant cells in atomic bomb
survivors (LANGLOIS
et al. 1987) provides a theoretical
equationfortheappearance
of preleukemic cells,
which are assumed to originate from a hemopoietic
stem cell heterozygous for a recessive oncogene, after
homozygosity through crossing over or a second recessive oncogenicmutation. T h e resultantdose-response equation approximates fairly well the actual
dose-response relationship of deaths from leukemia
among atomic bomb survivors (KONDO 1988).
We thank T . MAESHIMAfor encouraging the present work, R.
C. VON BORSTEL,
S. ABRAHAMSON,
R. MIKI,S. NAKAI,M. S. SASAKI,
and H. OCAWA for
helpful discussion and suggestions during preparation of the manuscript, Y. AOKIfor operating the nuclear reactor, and S. HISANACA and
A. TANESAKA
for preparing the manuscript. This work was supported by grants from the Ministry of
Education, Science and Culture ofJapan, the Foundation for Promotionof Cancer Research, theFoundationforPromotion
of
Private University Research, the US.-Japan Joint Environmental
Panel, and Kinki University.
LITERATURE CITED
ALBERTS,B., D. BRAY,J. LEWIS,M. RAFF,K. ROBERTSand J. D.
WATSON,1989 Molecular Biology of the Cell, 2nd Ed. Garland
Publishing, New York.
BAKER,B. S., A. T . C. CARPENTERandP. RIPOLL, 1978 The
utilization during mitotic cell division of loci controlling meiotic
recombinationand
disjunction in Drosophila
melanogaster.
Genetics 90: 531-578.
BECKER,H.J.,1976
Mitotic recombination,pp1019-1087,
in
The Geneticsand Biology of Drosophila, Vol. IC, edited by M.
ASHBURNER
and E. NOVITSKI.Academic Press, New York.
BENDER,M. A,, 1970 Neutron-induced genetic effects: a review.
Radiat. Bot. 10: 225-247.
BOYD,J. B., and R. B. SETLOW,1976 Characterization of postreplication repair in mutagen-sensitive strains of Drosophila melanogaster. Genetics 84: 507-526.
BRENNER,
D. A,, A. C. SMICOCKIand R. D. CAMERINI-OTERO,
1986 Double-strand gap repair results in homologous recombination in mouse L cells. Proc. Natl. Acad. Sci. USA 83:
1762-1766.
CARPENTER,
A. T . C., 1984 Meiotic roles of crossing-over and of
gene conversion. Cold Spring Harbor Symp. Quant. Biol. 4 9
23-29.
CATCHESIDE,
D. G., 1977 The Genetics of Recombination. University
Park Press, Baltimore.
A , , G. H. BALLANTYNEand D. G. HOLM, 1971 Studies
CHOVNICK,
on gene conversion and its relationship to linked exchange in
Drosophila melanogaster. Genetics 69: 179-209.
CuR'rIs. I>., S. H.
CLARK, A. CHOVNICK
and W. BENDER,
1989 Molecular analysis of recombination events in Drosophila. Genetics 122: 653-66 1.
EDELMANN,
W., B. KROEGER,M. GOLLERand I. HORAK, 1989 A
recombination hotspot in the LTR ofa mouse retrotransposon
in an in vitro system. Cell 57: 937-946.
FRANKENBERG, D.
D.,T . GOODHEAD,
M. FRANKENBERC-SCHWACER,
R. HARBICH, D. A. BANCE and R. E. WILKINSON,
1986 Effectiveness of 1.5 keV aluminum K and0.3 keV
carbon K characteristic X-rays at inducing DNA double-strand
breaks in yeast cells. Int. J. Radiat. Biol. 5 0 727-741.
FRIEDBERC,
E. C., 1985 DNA Repair. W. H. Freeman, New York.
FUJIKAWA,
K., and S. KONDO,1986 DNA repair dependence of
somatic mutagenesis of transposon-caused white alleles in Drosophilamelanogaster aftertreatment with alkylating agents.
Genetics 112: 505-522.
GARCIA-BELLIDO,
A , , 1972 Some parameters of mitotic recombination in Drosophila melanogaster. Mol. Gen. Genet. 115: 5472.
GARCIA-BELLIDO,
A,, and J. DAPENA,1974Induction,detection
and characterization ofcell differentiation mutants in Drosophala. Mol. Gen. Genet. 1 2 8 117-130.
GARCIA-BELLIW,
A , , and J. R. MERRIAM,1971a Genetic analysis
of cell heredity in imaginal discs of Drosophilamelanogaster.
Proc. Natl. Acad. Sci. USA 68: 2222-2226.
GARCIA-BELLIDO,
A,, and J. R. MERRIAM,1971bParameters of
the wing imaginal discdevelopment ofDrosophila melanogaster.
Dev. Biol. 24: 61-87.
GARDNER,
E. J., 1983 Human Heredity. John Wiley & Sons, New
York.
GOODHEAD,
D. T., 1982 An assessment of the role of microdosimetry in radiobiology. Radiat. Res. 91: 45-76.
GRAF,U., F. E. WURCLER,A. J. KATZ, H. FREI, H. JUON, C. B.
HALL and P. G. KALE,1984 Somatic mutation and recombinationtest in Drosophilamelanogaster. Environ. Mutagen. 6:
153-188.
GRAF,U . , H. FREI, A.KAcI, A. J. KATZ and F. E. WURCLER,
1989 Thirty compounds tested in the Drosophila wing spot
test. Mutat. Res. 222: 359-373.
HASTINCS,P. J., 1988 Conversion events in fungi, pp. 397-428,
in Genetic Recombination, edited by R. K. LAPATIand R. SMITH.
American Society for Microbiology, Washington, D.C.
HAYNES, R. H., andB. A. KUNZ, 1981 DNA repair and mutagenesis in yeast, pp. 37 1-414 in The Molecular Biology of the Yeast
Saccharomyces: Lije Cycle and Inheritance, edited by J. STRATHERN, E. W. JONES and J. R. BROACH.Cold SpringHarbor
Laboratory, Cold Spring Harbor, New York.
HAYNIE,
J. L., and P. J. BRYANT, 1977 T h e effects of X-rays on
the proliferation dynamics of cells in the imaginal wing disc of
Drosophila melanogaster. Wilhelm Roux's Arch. 183: 85-100.
HILLIKER,
A. J., and R. APPELS,1989 The arrangement of interphase chromosomes:structuraland
functionalaspects. Exp.
Cell Res. 185: 297-318.
HILLIKER,A., and A. CHOVNICK,1981Further observations on
intragenicrecombination in Drosophilamelanogaster. Genet.
Res. 38: 281-296.
HOLT, P. D., 1988 Biophysical interpretation of the response of
Chinese hamster cells to 24 keV neutrons. Br. J. Radiol. 61:
1142-1 146.
T. TSUJIMURA,
T . KURODA,M. KAWAMI
HOSHI,M., S. TAKEOKA,
and S. SAWADA, 1988 Dosimetricevaluation of 252Cfbeam
for use in radiobiology studies at Hiroshima University. Phys.
Med. Biol. 33: 473-480.
K. K., H. D. KLINCERand F. H. RUDDLE,1989 Human
KENNETH,
gene mapping 10. Cytogenet. Cell Genet. 51: 1-1 148.
J. A., and P. RIPOLL,1981 Spontaneous mitotic recomKENNISON,
bination and evidence for an X-ray-inducible system for the
repair of DNA damage in Drosophila m e h o g a s t e r . Genetics 98:
91-103.
Recombination
Mitotic
KNUDSON,A. G., JR., 1986 Genetics of human cancer. Annu. Rev.
Genet. 2 0 23 1-251.
KONDO, S . , 1988 Mutation and cancer in relation to the atomicbomb radiation effects. Jpn. J. Cancer Res. (Gann) 7 9 785799.
KONDO. S., 1989 The mechanism of mutation in relation to evolution and radiation (inJapanese). Jpn.J. Genet. 64: 137-163.
KOVACS,G., L. WILKENS
and T. PAPP, 1988 Nondisjunction reduplication of chromosome 3 is not a common mechanism in
the development of human renal cell tumors. Cytogenet. Cell
Genet. 48: 242-243.
KUNZ, B. A,, and R. H. HAYNES,
1981 Phenomenology and genetic control of mitotic recombination inyeast. Annu. Rev.
Genet. 15: 57-89.
KYOIZUMI,S., N. NAKAMURA,
M. HAKODA,
A. A. AWA,M. A. BEAN,
R. H. JENSEN and M. AKIYAMA,
1989a Detection of somatic
mutations at the glycophorin A locus in erythrocytes of atomic
bomb survivors using a single beam flow sorter. Cancer Res.
49: 581-588.
KYOIZUMI,
S., N. NAKAMURA,
H. TAKEBE,
K. TATSUMI,
J. GERMAN
and M. AKIYAMA,
198913 Frequency of variant erythrocytes
at the glycophorin-A locus in two Bloom’s syndrome patients.
Mutat. Res. 214: 215-222.
LANGLOIS,
R. G . , W. I.. BIGBEE,S. KYOIZUMI,
N. NAKAMURA,
M.
A. BEAN,M. AKIYAMA
and R. H. JENSEN, 1987 Evidence for
increased somatic cell mutations at the glycophorin A locus in
atomic bomb survivors. Science 2 3 6 445-448.
LANGLOIS,
R. G., W. L. BIGBEE,R. H. JENSEN and J. GERMAN,
1989 Evidence for increased invivo mutation and somatic
recombination i n Bloom’s syndrome. Proc. Natl. Acad.Sci.
USA 86: 670-674.
LEENTHOUTS,
H. P., and K. H. CHADWICK, 1978The crucial role
of DNA double-strand breaks in cellular radiobiological effects.
Adv. Radiat. Biol. 7: 55-101.
LEWIS,E. B., 1969 Cytological location of the multiple-wing-hairs
(mwh) gene in Drosophila melanogaster. Drosophila Inform.
Serv. 4 4 188.
LINDSLEY,
D. L., and E. H. GRELL,1968 GeneticVariations of
Drosophila melanogaster. Carnegie Inst. Wash. Publ. 627.
MANNEY,T. R., and R. K. MORTIMER,
1964 Allelic mapping in
yeast by X-ray-induced mitotic reversion. Science 143: 581582.
MERRIAM,J. R., and A. GARCIA-BELLIDO,
1972 A model for
somatic pairing derived from somatic crossing over with third
chromosome rearrangements in Drosophila melanogaster. Mol.
Gen. Genet. 115: 302-313.
METZ, C. W., 1916 Chromosome studies on the Diptera. 11. The
paired association of chromosomes in the Diptera and its significance.J. Exp. Zool. 21: 213-280.
MORTIMER,R., T . BRUSTAD
and D.V. CORMACK,
1965 Influence
of linear energy transfer and oxygen tension on the effectiveness of ionizing radiations for induction of mutations and
lethality in Saccharomyces cerevisiae. Radiat. Res. 2 6 465-482.
MULLER,J. H., 1946 Production of mutations. In Nobel LecturesPhysiology or Medicine. Published for the Nobel Foundation by
Elsevier, New York.
NAKAI,S., and S. MATSUMOTO, 1967 Two types of radiation
sensitive mutants in yeast. Mutat. Res. 4: 129-136.
NAKAI,
S., and R. MORTIMER, 1967 Induction of different classes
of genetic effects in yeast using heavy ions. Radiat. Res. Suppl.
7: 172-181.
NAKAI,
S., and R. MORTIMER,1969 Studies of the genetic mechanism of radiation-induced mitotic segregation in yeast. Mol.
Gen. Genet. 103: 329-338.
O’BRIEN,S. J., 1987 Genetic Maps, Vol.4. Cold Spring Harbor
Laboratory, Cold Spring Harbor, N.Y.
ORR-WEAVER, T. L., and J. W. SZOSTAK,
1985 Fungal recombination. Microbiol. Rev. 4 9 33-58.
in Drosophila
165
PEAK,M. J., J. G. PEAKand E. R. BLAZEK,1988 Symposium
report: radiation-induced DNA damage and repair (Argonne
National Laboratory Symposium, 1988). Int. J. Radiat. Biol.
54: 513-519.
POSTLETHWAIT,
J. H., 1978 Clonal analysisofDrosophila cuticular
patterns, pp. 359-441 in The Genetics and Biology of Drosophila,
Vol. 2c, edited byM. ASHBURNER
and T. R. F. WRIGHT.
Academic Press, New York.
RAJU, M. R., U. GNANAPURANI,
B. STACKLER,
B. I. MARTINS, U.
MADHVANNATH,
J. HOWARD,
J. T. LYMAN and
R. K. MORTIMER, 1971 Induction of heteroallelic reversions and lethality
in Saccharomyces cerevisiae exposed to radiations of various LET
(“’Co y-rays, heavy ions, and T - mesons) in air and nitrogen
atmospheres. Radiat. Res. 47: 635-643.
RAY,A., N. MACHIN and F.W. STAHL,1989 A DNA double
chain break stimulates triparental recombination in Saccharomyces cereuisiae. Proc. Natl. Acad. Sci. USA 86: 6225-6229.
RESNICK,M. A., 1976 The repair of double-strand breaks in DNA:
a model involving recombination. J. Theor. Biol. 59: 97-106.
RUBIN,J. S., 1988 The molecular genetics of the incision step in
the DNA excision repair process. Int. J. Radiat. Biol. 54: 309365.
RYO,H., and S. KONDO,1986 Photoreactivation rescue and hypermutability of ultraviolet-irradiated excisionless Drosophila
melanogaster larvae. Proc. Natl. Acad.Sci. USA 83: 33663370.
SAEKI,T., 1. MACHIDAand S. NAKAI, 1980 Genetic control of
diploid recovery after y-irradiation in the yeast Saccharomyces
cereuisiae. Mutat. Res. 73: 251-265.
SASAKI,
M. S., K. KOBAYASHI,
K. HIEDA,T . YAMADA,
Y . EJIMA,H.
MAEZAWA,Y. FURUSAWA,
T . ITO and S. OKADA,1989
Induction of chromosome aberrations in human lymphocytes
by monochromatic X-rays of quantum energy between 4.8 and
14.6 keV. Int. J. Radiat. Biol. 5 6 975-988.
SAVAGE,
J. R. K., 1989 The production of chromosome structural
changes by radiation: an update of LEA (1946), Chapter VI.
Br. J. Radiol. 62: 507-520.
SCHINZEL,
A., 1984 Catalogue of Unbalanced Chromosome Aberrations in Man. Walter de Gruyter, Berlin.
SCRABLE,
H. J., D. P. W I ~ EB., C. LAMPKIN
and W. K. CAVENEE,
1987 Chromosomal localization
of
human rhabdomyosarcoma locus by mitotic recombination mapping. Nature
329: 645-647.
STERN,C., 1936 Somatic crossing over and segregation in Drosophila melanogaster. Genetics 21: 625-730.
STERN,C., 1969 Somatic recombination within the white locus of
Drosophila melanogaster. Genetics 62: 573-58 1.
THALER,
D. S., and F. W. STAHL,1988 DNA double-chain breaks
in recombination of phage and of yeast. Annu. Rev. Genet. 22:
169-197.
THOMAS,
B. J., and R. ROTHSTEIN,
1989 Elevated recombination
rates in transcriptionally active DNA. Cell 56: 619-630.
TOKUNAGA,
C., 1973Intragenic
somatic recombination in the
lozenge cistron of Drosophila. Mol. Gen. Genet. 125: 109-1 18.
UNRAU,P., 1986 The relative biological effectiveness of 14.5MeV neutrons for the induction of gene conversion and mutation in yeast. Radiat. Res. 107: 39-48.
UNRAU,
P., 1987 The RBE of neutrons for induced mitotic gene
conversion in “error-prone repair” defective yeast. Radiat. Res.
111: 92-100.
VOELKEL-MEIMAN,
K.,R.L.
KEIL and G. S. ROEDER, 1987
Recombination-stimulating sequences in yeast ribosomal DNA
polymerase I. Cell 48: 1071-1079.
WESTON,A., J. C. WILLEY,R. MODALI,H. SUGIMURA,
E. M.
MCDOWELL,
J. RESAU,B. LIGHT,A. HAUGEN,
D. L. MANN,B.
F. TRUMP
and C. C. HARRIS,1989 Differential DNAsequence deletions from chromosomes 3, 11, 13,and 17 in
squamous-cell carcinoma, large-cell carcinoma, and adenocar-
166
T. Ayaki et al.
cinotn;) of the human lung. Proc. Natl. Acad.Sci. USA 8 6
5099-5 103.
WILDENBERG,
J., 1970 The relation of mitotic recombination to
DNA replication i n yeast pedigrees. Genetics 6 6 291-304.
WITKIN,E. M . , 1976 Ultraviolet mutagenesisand inducible DNA
repair in Escherichia coli. Bacteriol. Rev. 4 0 869-907.
YASUBUCHI,
S.,M. HOSHI,T. ITOH, S. HISANAGA,T. NIWA, R.
MIKIand S. KONDO,1989 Dosimetry
fast
of
neutrons in 1 W
nuclear reactor withplastic nuclear-track detectors. Radioisotopes 38: 359-365.
Yoo, M. A,, H. Ryoand S. KONDO, 1985 Differential hypersensitivities of Drosophila melanogaster strains with mei-9, mei-41
and mei-9 mei-41 alleles to somatic chromosome mutations after
larval X-irradiation. Mutat. Res. 1 4 6 257-264.
Communicating editor: A. CHOVNICK