INT. J . RADIAT. BIOL ., 1995, VOL . 68, NO. 2, 177-183

Dose-response of a radiation induction of a germline mutation ata hypervariable mouse minisatellite locus

Y. J. FANt, Z . WANGt, S . SADAMOTO+, Y. NINOMIYAt, N. KOTOMURAt,K. KAMIYAt, K. DOHI+, R. KOMINAMI§ and O. NIWA*t

(Received 23 January 1995 ; revision received 3 April 1995; accepted 5 April 1995)

Abstract . Dose-response of an induction of a germline mutationwas studied at a hypervariable mouse minisatellite locus, Ms6hm,which consists of tandem repeats of a sequence motif GGGCA.Male C3H/HeN mice were exposed to various doses of 60Co y-rayand mated with unirradiated C57BL/6N female mice . Matingswere done at various time after irradiation to assess the effects ofradiation on spermatozoa, spermatids and spermatogonia . DNAsamples of F, offsprings were analysed by Southern blotting for therepeat length mutation at the Ms6hm locus. The mutationfrequency per gamete of the paternal allele was 9-1% for theunirradiated control group. The spermatids stage was most sensitiveto radiation and a statistically significant dose-response wasobserved. The mutation frequency of the paternal allele in F, miceincreased to 22% for 1 Gy, 28°/o for 2 Gy, and 28% for 3 Gy . Thespermatogonia stage was less sensitive to radiation, and the mutationfrequencies of the paternal allele were 14% for 2 Gy, and 16% for3 Gy. The spermatozoa stage germ cells were also less sensitive andthe frequency of mutation of the paternal allele increased to 14%for 3 Gy. However, these increases were statistically not significant .Possible mechanisms of radiation induction of germline mutation atthe hypervariable minisatellite locus will be discussed.

1. Introduction

Analysis of germline mutation by ionizing radiationis important for the risk evaluation of the public and hasbeen the subject of intensive studies. Germline muta-tions among human population are difficult to examinebecause of a number of difficulties such as : geneticheterogeneity in human populations, and the lowfrequencies of germline mutation at single copy genes .A study conducted on the second generation of theA-bomb survivors in Hiroshima and Nagasaki revealed

*Author for correspondence .Department of Molecular Pathology, Research Institute for

Radiation Biology and Medicine, Hiroshima University, Minami-ku, Hiroshima 734, Japan.

Second Department of Surgery, Hiroshima University School ofMedicine, Minami-ku, Hiroshima 734, Japan .

§Department of Biochemistry, Niigata University School ofMedicine, Asahimachi-dori, Niigata 951, Japan .

0955-3002/95 $10.00 © 1995 Taylor & Francis Ltd.

no significant increase in germline mutations (Neel et al.1990). In mouse experiments, the specific locus test hasbeen utilized to analyse radiation-induced mutation(Russell 1951). Because of the low frequency ofgermlinemutation at single copy genes, this analysis requires alarge number of mice (tens of thousands) for a reliableestimation of mutation induced in germ cells (Russelland Kelly 1982a) . Nevertheless, the specific locus testhas been applied to assess radiation-induction ofgermline mutation in relation with dose, dose-rate, andgerm cell stages in each (Russell et al. 1958, Russell1977, Russell and Kelly 1982a, b) .Recent analysis of human and mouse genomes

revealed a variety of sequence elements that are subjectto much higher frequency of mutation (Jeffreys 1987,Jeffreys et al. 1987, 1988) . Because of high frequenciesof spontaneous germline mutation at these loci, theymay provide a more efficient marker to monitor thegenetic effect of radiation . Indeed, DNA fingerprintanalysis revealed that irradiation of mouse germ cellsof the spermatogonia stage with 0 . 5 or 1 Gy y-raysresulted in an increase in germline mutation at mouseminisatellite loci among the offspring (Dubrova et al.1993). We also studied germline mutation at the mouseminisatellite locus Ms6hm and showed that the mu-tation rate can be increased significantly by irradiationof spermatid cells (Sadamoto et al. 1994). The inducedmutation frequency at the locus was in the order of 10 -1for a dose of 3 Gy and, therefore, appears to be too highto be accounted for by the direct action of DNAdamages induced by radiation . A similar high frequencywas reported for the germline-transmissible mutationthat induces tumours in the next generation (Nomura1982). The high frequency of these mutations can onlybe explained by an indirect mechanism of mutationinduction triggered by radiation .

In this paper we have analysed the dose-response ofmutation induction at the Ms6hm locus after irradiationof cells at three stages of spermatogenesis . The results

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

cgill

Uni

vers

ity o

n 11

/27/

14Fo

r pe

rson

al u

se o

nly.

178

confirm our previous findings. In addition, a cleardose-response was observed in the mutation frequen-cies for spermatid irradiation .

2. Materials and methods

2.1 . Mice

Female C57BL/6N and male C3H/He mice werepurchased from Charles River Japan Inc . They werehoused in our animal facility and fed with commercialpellets and tap water ad libitum, under the condition ofconstant temperature (24°C) and a regular 12-hlight/dark cycle .

2.2 . Irradiation and mating

The experimental design was essentially the same asthat published previously (Sadamoto et al. 1994). Thebreeding of the unirradiated control group was firstmade in which 8-10-week-old C3H/He males weremated with C57BL/6N females of similar age . As forthe irradiated groups, C3H/He males were exposed to1, 2 or 3 Gy y-rays at a dose-rate of 0 .5 Gy/min usingthe 60Co source at our institute . They were mated withC57BL/6N females immediately, 3 weeks, or 11 weeksafter irradiation for analysis of radiation effects on thespermatozoa, spermatids or spermatogonia stage germcells .

2.3 . Southern blotting analysis

DNA was extracted from tails of F, mice as describedpreviously (Sadamoto 1994) . Briefly, tails were cut,minced and solubilized in a buffer containing0 . 1 M NaCl, 5 mm EDTA, 20 mm Tris-HCl, pH 8.0,1% sodium dodecyl sulphate (SDS), 100 pg/ml RNaseA and 100 pg/ml proteinase K. After digestion at 50°Covernight, DNA was extracted with phenol-chloroformand recovered by ethanol precipitation. DNA wasdigested with Hae III and 4 jig of which was separatedby electrophoresis through 20-cm 0 .7% agarose gel at1 V/cm for 24 h in 1 X TBE (Tris base 89 mm, boricacid 89 mm, EDTA 2 mm) . DNA was then transferredto Nylon membrane filters (Southern 1975) . The filterswere prehybridized in 5 X SSC, 1% SDS at 65°C for2 h and then hybridized in the same buffer containingthe 32P-labelled probe at 65°C for 16-24 h . The filterswere washed with 0 .01 X SSC at 65 °C for 30 min andexposed to X-ray film .

Y. 3. Fan et al .

2.4. Minisatellite probe

The minisatellite probe Pc-1 is a locus-specific cloneisolated from the mouse genome by hybridization to theMo-1 minisatellite probe (Mitani et al. 1990). Thesequence analysis revealed that it was identical to theMs6hm locus isolated by others (Kelly et al. 1989). ThePc-1 consist of tandem repeats of a GGGCA motif andis highly polymorphic among inbred mouse strains .

2.5 . Assembly and analysis of data

As for 3-Gy dose points, data of our previous work(Sadamoto et al. 1994) were included to obtain a largerand more reliable set of data . The mutation frequencieswere examined for statistical significance using theChi-square test .

3. Results

3.1 . Scoring of mutation at the Ms6hm locus

Parental mice were first screened for the heterozygos-ity of the Ms6hm locus using the Pc-1 probe . C3H/He,and C57BL/6N strains carried the Ms6hm locus ofabout 3 . 7 and 8 . 3 kb respectively . The hypervariablenature of the locus was evident from the fact that theband length varied frequently even within the strain . Inorder to avoid complication in the analysis of DNA ofF, mice, parental mice carrying more than one Pc-lband, an indication of the heterozygosity, were ex-cluded from the present study .

The majority of germline mutation at the Ms6hmlocus is due to variations in the number of the repeat,which lead to changes in the band length in theSouthern analysis. Some human minisatellite lociconsist of a stretch of DNA in which repeat units ofslightly different sequences are intermingled . For theseloci, a digital approach of scoring the mutation ispossible (Jeffreys et al. 1991). The mouse Ms6hmconsists of a simple repeat of a sequence motif and thedigital approach of detection of mutation was notpossible . Therefore, we were required to score for a shiftin mobility in Southern analysis as a mutation of theMs6hm locus. We applied a 2% band shift as a criterionof mutation (as in our previous study, Sadamoto et al.1994). Briefly, F, mice were scored as mutant either oftheir two bands shifted in their electrophoretic mobilityby > 2% in distance from the corresponding parentalband. Examples of F, mice carrying the decrease inlength type mutation on the C3H/HeN allele and theincrease in length type mutation on the C57BL/6Nallele are shown in Figure 1 .

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

cgill

Uni

vers

ity o

n 11

/27/

14Fo

r pe

rson

al u

se o

nly.

C57BL 10-

C3H 01`

Radiation dose-response of minisatellite germline mutation

179

P/M 1

A

2 P/M

The total mutation frequency was obtained bydividing the number of the mutant alleles by the totalnumber of alleles analysed . The mutation frequencieswere also scored for the paternal and the maternalalleles, separately.

3.2 . Background mutation frequency of the control group

The frequency of germline mutation at the Ms6hmlocus was analysed for three stages of spermatogenesis ;spermatozoa, spermatids and spermatogonia . Table 1and Figure 2 summarize the results of the analysis . Asfor the unirradiated control group, 131 F l mice wereanalysed that were born to the matings of 14 males and22 females . The total mutation frequency of this groupwas 10%. The mutation frequencies of the maleC3H/HeN and female C57/BL/6N alleles were 9 . 1and 12% respectively . Among these mutations, theincrease- and the decrease-in-length types were ob-served at similar frequencies (Table 1) .

C57BL W-

C3H

B

P/M 1 2 P/M

Figure 1 . Mutation of the Ms6hm locus at the paternal and the maternal allele . (A) A litter of F 1 mice born to 2-Gy spermatids irradiationgroup was analysed . P/M is a mixture of DNA from both male and female parents, and 1 and 2 are two F, mice born to these parents .Note that the F1 mouse of number 2 carries mutation at the paternal allele . (B) A litter of F, mice born to 1-Gy spermatids irradiationgroup was analyzed . Note that the F1 mouse of number 2 carries mutation at the maternal allele .

3.3 . Irradiation of the spermatozoa stage germ cells

One dose point of 3 Gy was tested for the spermato-zoa stage irradiation that involved 182 F, mice born tothe immediate matings of 28 irradiated males and 35females. The total mutation frequency increased butnot significantly from 10% of the control level to 15% .The paternal mutation frequency increased from 9 . 1of the control level to 14% . The mutation frequency ofthe maternal allele, which derived from unirradiatedpartners, also increased from 12 to 15% . The increasein length-type mutation was observed more frequentlyin male and female alleles of F l mice (Table 1). Themutation frequencies of the paternal and the maternalalleles are plotted in Figure 2 . It may require a largerscale experiment to determine if length changes can beinduced in the spermatozoa stage .

3 .4 . Irradiation of the spermatids stage germ cells

Three dose points were tested for the effect ofradiation on the spermatids stage germ cells . A total of

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

cgill

Uni

vers

ity o

n 11

/27/

14Fo

r pe

rson

al u

se o

nly.

180

eP

Ucd0tr5-Lc0..+

32

30

20

10

0

0

0

1

181 F, mice were analysed for the 1-Gy dose point,which were born to the 3-week matings of 18 irradiatedmales and 30 females . The total mutation frequencyincreased to 16%. The mutation frequency of thepaternal allele increased to 22% while the maternalallele stayed at 9 . 9% of the control level

A total of 82 F, mice were analysed for the 2-Gy dosepoint, which were born to the matings between 12irradiated males and 16 females . The total mutationfrequency increased to 20% . The mutation frequencyof the paternal allele was 28% and the mutationfrequency of the maternal allele stayed at 12% .

Irradiation of the spermatids stage germ cells by 3 Gydecreased the litter size of the offsprings significantly .Analysis of the 3-Gy dose point involved 104 F 1 miceborn to the matings of 25 irradiated males and 25females. The total mutation frequency was 20% inwhich the paternal allele mutation frequency increasedto 28% while the maternal allele stayed at 12% .

The increases in the mutation frequencies of thepaternal allele were statistically significant for all dose

Y. J. Fan et al .

1 I I

2

spermatid paternal allele

spermatogonia paternal allele

Aspermatozoa maternal allelespermatozoa paternal allele

0 spermatid maternal allele

0 spermatogonia maternal allele

3

Radiation dose (Gy)

Figure 2 . Dose-response of mutation induction . Mutation frequencies of Table 1 are plotted against doses of y-ray . Open symbols, thematernal allele mutation ; closed symbols, the paternal allele mutation ; triangles, spermatozoa irradiation ; squares, spermatidsirradiation ; and circles, spermatogonia irradiation . Points for the spermatozoa irradiation were not connected with line because theyconsist of only one dose points.

points of the spermatid irradiation . Interestingly theincrease-in-length-type mutation predominated themutation of the paternal allele . The mutation frequen-cies of the spermatids stage groups were plotted againstdose of radiation (Figure 2) . It is likely that the mutationfrequency of the paternal allele increased linearly up to2 Gy where the curve reached a plateau .

3 .5 . Irradiation of the spermatogonia stage germ cells

Two dose points of 2 and 3 Gy were analysed for thespermatogonia stage germ cells . A total of 133 F1 micewere born to the 11 weeks matings of 11 irradiatedmales and 17 females . The total mutation frequency,9 . 7%, did not show a sign of radiation effect . However,the mutation frequency of the paternal allele was 14%and that of the maternal allele 6% . As for the 3-Gyirradiation, 95 F 1 mice were analysed and the totalmutation frequency was 11 % in which the paternal

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

cgill

Uni

vers

ity o

n 11

/27/

14Fo

r pe

rson

al u

se o

nly.

Radiation dose-response of minisatellite germline mutation

Table 1 . Dose-reponse of Pc-1 mutation among F, born to irradiated the C3H male mouse .

ad and i denote decrease-in-length and increase-in-length mutation respectively .by< 0 . 05 (compared with C3H control) .°p < 0 . 005 (compared with C3H control) .dp < 0 . 005 (compared with C3H control) .

allele mutation was 16% while that of the maternalallele 7 . 3%. The mutation frequencies of the maternalallele for spermatogonia irradiation were always lowerthan that of the control group . However, these numberswere statistically not significant . The data points wereplotted in Figure 2 .

4. Discussion

4.1 . Dose-response and stage sensitivity

Our present study demonstrates that the spermatidsstage germ cells are quite sensitive to y-irradiation forthe induction ofgermline mutation at the Ms6hm locus .The dose-response curve indicated that the mutationfrequency of the paternal allele increased linearly up to2 Gy where the curve reached a plateau . The sperma-tozoa and spermatogonia stages were less sensitive toradiation induction of the germline mutation at theMs6hm locus . The mutation frequencies of the paternalallele for these stages increased only to 14 and 16% atthe dose of 3 Gy respectively . Interestingly, the mu-tation frequency of the maternal allele also seems to beelevated among 182 Fi mice born to the spermatozoairradiation . A similar tendency of the increase of thematernal allele mutation was noted previously duringanalysis of 77 F, mice (Sadamoto et al. 1994). A largernumber of F, mice should be analysed to verify thestatistical significance of these values .

The doubling doses for the germline mutation of thepaternal allele of the Ms6hm locus can be calculatedfrom the present results, although only the spermatids

181

stage data survives the statistical testing . These are 6,0 .79 and 6 Gy for the spermatozoa, spermatids andspermatogonia stages respectively. They are somewhatlarger than that for the specific locus mutation at thespermatogonia stage (UNSCEAR 1993b) .

4.2 . High sensitivity of the spermatids stage gerrn cells

A high sensitivity to radiation of the spermatids stagegerm cells has been well documented for severalparameters. These include specific locus mutation(Searle 1974), dominant lethals (Ehling 1971), malfor-mation and tumour development in the secondgeneration (Nomura 1982). Our present study demon-strates that the minisatellite mutation is an additionalmarker that is sensitive to ionizing radiation at thespermatid stage . The mechanism of this high sensitivityis not known at present . Drastic changes of chromatinstructure such as euchromatic state in the spermatocytenucleus to heterochromatic state in the sperm head maybe responsible for this high sensitivity of the spermatidsstage germ cells . Radioresistance of the spermatogoniastage is thought to be due to its high capacity to repairDNA damages in this stage of sperm cells . Reductionof mutation frequency upon fractionation and lowdose-rate irradiation demonstrates the presence ofrepair system in the spermatogonia stage germ cells(Russell and Kelly 1982a, b). However, the apparentradioresistance of spermatogonia cells may also beexplained by selection against severely damaged cells toenter and undergo successful meiosis .

Group

Number Mutants (%)

Male Female F1 Total Paternal Maternal

Control 14 22 131 27 (10%) 12 (9-1%, 4d + 8i)a 15 (12%, 7d + 8i)

Spermatozoa irradiation (Gy)3 28 35 182 54(15%) 26 (14%, 5d+21i) 28 (15%, 11 d + 17i)

Spermatids irradiation (Gy)1 18 30 181 58(16%) 40 (22%, 2d + 38i)b 18 (9 . 9%, 7d+ lli)2 12 16 82 33(20%) 23 (28%, 6d + 17i)` 10 (12%, 6d + 4i)3 25 25 104 41(20%) 29 (28%, 4d + 25i)d 12 (12%, 4d + 8i)

Spermatogonia irradiation (Gy)2 11 17 133 26(9 , 7'/.) 18 (14%, 9d + 9i) 8 (6 . 0%, 2d + 6i)3 12 13 95 22 (11%) 15 (16%, 2d+ 13i) 7 (7 . 3%, 2d + 5i)

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

cgill

Uni

vers

ity o

n 11

/27/

14Fo

r pe

rson

al u

se o

nly.

182

Y. j. Fan et al.

4.3 . Indirect mechanism of mutation induction at the Ms6hmlocus

As has been pointed out, the frequencies of mutationat the Ms6hm locus observed in our study are at leasttwo orders of magnitude higher than the number ofDNA damages induced by radiation (Sadamoto et al.1994). One single strand break occurs every 3000 kbDNA at 1 Gy y-rays (UNSCEAR 1993a). The size ofthe paternal allele of the Ms6hm locus is 3 kb and theexpected mutation frequency is in the range of 10- 3 atthis dose . Therefore, DNA damage is not the directcause of the germline mutation at the Ms6hm locus . Wepostulate that radiation first triggers instability of thegenome, which then operates on this hypersensitivelocus to change the repeat number .

4.4 . Postmeiotic mutation

The length variations in microsatellite sequences arethought to occur during DNA replication throughfailure of correction of slippage of template and newlysynthesized strands (Strand et al. 1993). Mismatchrepair genes involved in the correction have beencloned and the mutation of these genes was shown tobe involved in the development of human tumours(Parsons et al. 1993, Papadopoulos et al. 1994). Incontrast with mutation of microsatellite sequences, thelength type mutation of minisatellite sequences wasproposed to occur through gene conversion or recom-bination between homologous alleles (Monckton et al.1994) . These processes require at least two copies of theallele to exchange the sequence information .

We have observed the mutation in F 1 mice when thespermatogonia stage cells are irradiated . This stage ofgerm cells carry both maternal and paternal alleles inthe same nucleus and exchange of the sequenceinformation is possible . However, we observed that thespermatids stage germ cells are the most vulnerable toradiation induction of mutation at the Ms6hm locus .Germ cells of this stage are post-meiotic and carry onlyone copy of the gene . Thus, the mutation at this stagemay be induced by a mechanism unrelated to usualgene conversion or homologous recombination . Alter-natively, it is possible that heteroduplex formation mayoccur in the spermatocyte stage germ cells andirradiation simply enhances the rate of gene conversionin the spermatid stage germ cells . Replication slippagecannot account for the mutation at the Ms6hm locus inthe spermatid stage germ cells where DNA replicationdoes not take place .

Spermatids stage germ cells still retain some of theDNA-metabolizing activities. Unscheduled DNA syn-thesis, or repair replication, was detected in the

spermatid stage germ cells after treatment with methyl-methenesulphonate and X-ray (Sotomayor et al. 1979,Inoue et al. 1993). Therefore, the germline mutation ofthe Ms6hm locus at the spermatids stage germ cellssomehow has to occur through this repair replicationprocess. The replication slippage may well take placeduring this repair process .

4 .5 . Spermatozoa irradiation

The chromatin of the spermatozoa stage germ cellsis tightly packed and the cells in this stage are thoughtto lack any of the DNA-metabolizing activities (Soto-mayor et al. 1979, Kato and Tanaka 1980) . Thissuggests that the germline mutation of the Ms6hm locusmust be occurring after the entry of the irradiated sperminto the oocytes . In the one cell-stage embryo, the firstDNA replication takes place separately in male andfemale nuclei . Damaged DNA carried by the spermmay activate gene functions that in turn trigger geneticinstability. If the induction of genetic instability involvescytoplasmic events such as synthesis of new proteins, itis reasonable to assume that the instability operates cisto the paternal allele as well as trans to the maternalallele of the locus. We still have to analyse a largernumber of Fl mice to obtain any statistically significantdata on the maternal allele mutation for spermatozoairradiation. Nevertheless, the observed increase of themutation at the maternal allele after irradiation of thespermatozoa stage germ cells is consistent with theproposed hypothesis .

Acknowledgements

We thank Drs M. S. Sasaki, Seymor Abrahamsonand James V. Neel for valuable discussions and M .Inoue for information on the repair activities duringspermatogenesis . We also thank Ms A. Kinomura andMs K. Mizuno for their excellent technical assistance,Mr T. Nishioka for photographic works and Ms T .Matsuura for typing the manuscript . This work wassupported by a Grant-in-Aid for Cancer Research fromthe Ministry of Education, Science and Culture, andgrants from the Ministry of Health and Welfare, fromthe Nuclear Safety Research Association, Tokyo, andfrom the Health Research Foundation, Kyoto, Japan .

References

DUBROVA, Y. E ., JEFFREYS, A. J . and MALASHENKO, A. M., 1993,Mouse minisatellite mutations induced by ionizing radi-ation . Nature Genetics, 5, 92-94 .

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

cgill

Uni

vers

ity o

n 11

/27/

14Fo

r pe

rson

al u

se o

nly.

EHLING, U . H., 1971, Comparison of radiation- and chemicallyinduced dominant lethal mutations in male mice . MutationResearch, 11, 35-44 .

INOUE, M., KURIHARA, T ., YAMASHITA, M. and TATSUMI, K., 1993,Effects of treatment with methylmethenesulfonate duringmeiotic and postmeiotic stages and maturation of spermato-zoa in mice . Mutation Research, 249, 179-186.

JEFFREYS, A. J., 1987, Highly variable minisatellites and DNAfingerprints . Biochemical Society Transactions, 15, 309-317 .

JEFFREYS, A. J., MACLEOD, A., TAMAKI, K., NEIL, D. L. andMONCKTON, D. G., 1991, Minisatellite repeat coding as adigital approach to DNA typing. Nature, 354, 204-209 .

JEFFREYS, A. J., ROYLE, N. J., WILsov, V. and WONG, I ., 1988 .Spontaneous mutation rates to new length alleles at tandemrepetitive hypervariable loci in human DNA . Nature, 322,278-281 .

JEFFREYS, A . J ., WILSON, V., KELLY, R., TAYLOR, B . A. and BUL-FIELD, G ., 1987, Mouse DNA `fingerprints' : analysis ofchromosome localization and germ-line stability of hyper-variable loci in recombinant inbred strains . Nucleic AcidsResearch, 15, 2823-2836 .

KATOH, M. and TAKADA, N ., 1980, Relationship between chromo-some aberrations in the first-cleavage metaphases andunscheduled DNA synthesis following paternal MMStreatment . Japanese Journal of Genetics, 55, 55-65 .

KELLY, R., BULFIELD, G., COLLICK, A., GIBBS, M. and JEFFREYS,A. J ., 1989, Characterization of a highly unstable mouseminisatellite locus : evidence for somatic mutation duringearly development . Genomics, 5, 844-856 .

MITANI, K., TAKAHASHI, Y. and KoMINAMI, R ., 1990, A GGCAGGmotif in minisatellite affecting their gem-dine instability .Journal of Biological Chemistry, 265, 15203-15210 .

MONCKTON, D . G., NEUMANN, R., GURAM, T., FRETWELL, N.,TAMAKI, K., MACLEOD, A. and JEFFREYS, A., 1994, Mini-satellite mutation rate variation associated with a flankingDNA sequence polymorphism . Nature Genetics, 8, 162-170 .

NEEL, J . V., SCHULL, W . J ., AWA, A. A., SATOH, C ., KATO, H.,OTAKE, M. and YOSHIMOTO, Y., 1990, The children ofparents exposed to atomic bombs : estimates of the geneticdoubling dose of radiation for humans . American Journal ofHuman Genetics, 46, 1053-1072.

NOMURA, T., 1982, Parental exposure to X-rays and chemicalsinduces heritable tumors and anomalies in mice . Nature, 296,575-577 .

PAPADOPOULOS, N., NICOLAIDES, N . C ., WEI, Y-F ., RUBEN, S. M.,CARTER, K . C., ROSEN, C . A., HASELTINE, W. A ., FLEISCH-MANN, R. D., FRASER, C . M., ADAMS, M . D., VENTER,J . C .,

Radiation dose-response of minisatellite germline mutation

183

HAMILTON, S . R., PETERSEN, G . M., WATSON, P ., LYNCH, H .T., PELTOMAKI, P., MECKLIN,J-P ., CHAPELLE, A ., KINZLER,K. W. and VOGELSTEIN, B., 1994, Mutation of mutL homo-log in hereditary colon cancer . Science, 263, 1625-1629 .

PARSONS, R ., LI, G-M., LONGLEY, M. J ., FANG, W-H., PAPADOPOU-LOS, N ., JEN, J ., DE LA CHAPELLE, A., KINZELER, K. W .,VOGELSTEIN, B. and MODRICH, P ., 1993, Hypermutabilityand mismatch repair deficiency in RER+ tumor cells . Cell,75, 1227-1236.

RUSSELL, W. L., 1951, X-ray-induced mutation in mice. Cold SpringHarbor Symposium on Quantitative Biology, 16, 327-336 .

RUSSELL, W . L ., 1977, Mutation frequencies in female mice and theestimation of genetic hazards of radiation in women .Proceedings of the National Academy of Sciences, USA, 74,3523-3527 .

RUSSELL, W. L. and KELLY, E . M., 1982a, Mutation frequencies inmale mice and the estimation of genetic hazards of radiationin men . Proceedings of the National Academy of Sciences, USA, 79,542-544 .

RUSSELL, W . L. and KELLY, E. M., 1982b, Specific-locus mutationfrequencies in mouse stem-cell spermatogonia at very lowradiation dose rates. Proceedings of the National Academy ofSciences, USA, 79, 539-541 .

RUSSELL, W. L ., RUSSELL, L . B . and KELLY, E. M., 1958, Radiationdose rate and mutation frequency . Science, 128, 1546-1550 .

SADAMOTO, S., SUZUKI, S., KAMIYA, K ., KOMINAMI, R ., DOHI, K.and NIwA, 0 ., 1994, Radiation induction of germlinemutation at a bypervariable mouse minisatellite locus .International Journal of Radiation Biology, 65, 549-557 .

SEARLE, A. G., 1974, Mutation induction in mice . Advances inRadiation Biology, 4, 131-207 .

SOTOMAYOR, R. E., SEGA, G . A. and CUMMING, R. B., 1979, Anautoradiographic study of unscheduled DNA synthesis in thegerm cells of male mice treated with X-ray and methylmethenesulfonate . Mutation Research, 62, 293-309 .

SOUTHERN, E . M., 1975, Detection of specific sequences amongDNA fragments separated by gel electrophoresis . Journal ofMolecular Biology, 98, 505-513 .

STRAND, M ., PROLLA, T. A., LISKAY, R. M. and PETES, T. D ., 1993,Destabilization of tracts of simple repetitive DNA in yeast bymutations. Affecting DNA mismatch repair. Nature, 365,274-276 .

UNSCEAR REPORT, 1993a, Annex F. Influence of Dose and Dose Rateon Stochastic Effect of Radiation (United Nations GeneralAssembly, Vienna), pp . 620-728 .

UNSCEAR REPORT, 1993b, Annex G. Hereditary Effects of Radiation(United Nations General Assembly, Vienna), pp. 729-804.

Int J

Rad

iat B

iol D

ownl

oade

d fr

om in

form

ahea

lthca

re.c

om b

y M

cgill

Uni

vers

ity o

n 11

/27/

14Fo

r pe

rson

al u

se o

nly.