Mutation Research, 192 (1987) 121-124 121 Elsevier

MTRL 032

Induction of sister-chromatid exchanges and exchange aberrations by UV light and quinacrine mustard in relation to chiasma formation in a

standard line and two oligochiasmatic mutants of Vicia faba L.

R i c h a r d V o g e l a n d B e r n d F r i e b e

Institut fiir Angewandte Genetik, Freie Universitiit Berlin, Albrecht-Thaer-Weg 6, D-IO00 Berlin 33 (West Germany)

(Accepted 18 May 1987)

Keywords: Sister-chromatid exchange; Quinacrine mustard; Chiasma formation; (Viciafaba L.).

Summary

The frequencies of sister-chromatid exchanges (SCE) and exchange aberrations (EA) in root tip cells were compared to chiasma format ion in pollen mother cells o f a standard line and two oligochiasmatic lines of Vicia f a b a L. SCE and EA were induced by UV light and quinacrine mustard. Between the lines SCE fre-

quencies were not different. The background level of SCE was doubled after UV irradiation and 4 times higher after exposure to quinacrine mustard in all Vicia lines analysed. However, the induced frequencies

of EA were found to be different under the same treatment conditions for the standard line and the oligo- chiasmatic mutants. Between the frequencies of induced EA and the frequencies of chiasmata a correlation

could be shown. The relationship between the formation of SCE and EA due to the reduced ability of meiotic recombina-

tion in the mutants of Vicia f a b a is discussed.

Sister-chromatid exchanges (SCE) as well as chromosomal aberrations can be induced by several chemical or physical agents. Recently, ex- periments with DNA endonucleases provided some evidence that the ultimate lesion for both events is a DNA double-strand break (Nowak and Obe,

1984; Obe et al., 1985; Natarajan et al., 1985). To complete format ion of SCE and chromosomal

Correspondence: Dr. Richard Vogel, Max von Pettenkofer- Institut des Bundesgesundheitsamtes, P.O. Box 330013, D-1000 Berlin 33 (West Germany).

aberrat ion of the exchange type (EA) reunion of the double-stranded DNA is necessary. In addi- tion, SCE and all types of chromosomal aberrat ion are visible endpoints of DNA damage. Therefore, SCE and EA seem to be related in format ion as well as in function as indicators of DNA damage. Another event which requires exchange of DNA duplices is the crossing-over (CO) of homologous chromosomes during prophase of the first meiotic division. However, CO occurs naturally as a mechanism to increase the individual variability of

the progenies. Notwithstanding the differences in function, the

0165-7992/87/$ 03.50 © 1987 Elsevier Science Publishers B.V. (Biomedical Division)

122

similarities in the formation process led us to study possible relationships between meiotic recombina- tion on one side and the endpoints of DNA damage (SCE and EA) on the other side. Therefore, SCE and EA induced by UV light and quinacrine mustard were determined in a standard line of Vicia faba L. and two mutants with step- wise reduced chiasma frequencies.

Materials and methods

Plant material Lateral root-tip meristems of a standard line

(var. 'Primus') and two mutants with reduced chiasma frequency were used in our experiments. The mutants had been obtained and described by Sj6din (1970). Linnert and coworkers (1981) again determined the chiasma frequencies analysing 100 pollen mother cells per line.

Mutagenic treatment Root tips were exposed either to 2.5 × 10 - 6 M

quinacrine mustard (Sigma Q-2000) for 60 min or to UV light (Osram 40 W/350 nm) at a distance of 30 mm for 60 min, which gives a dose of 5.97 J /cm 2.

SCE test

Before exposure to the mutagens root tips were grown in the presence of 5 '-bromodeoxyuridine (BrdU, 10 -4 M), fluorodeoxyuridine (FdU, 10 -7

M), uridine (Urd, 5 x 10 -6 M) during one cell cycle and during the second cycle in the presence of thymidine (Thd, 10 -4 M), Urd (5 x 10 -6 M). Final-

ly, root tips were fixed in ethanol and acetic acid (3 : 1). For sister-chromatid differentiation the flu- orescence-plus-Giemsa (FPG) staining technique has been described previously (Friebe, 1979). SCE were scored in 25 complete metaphases per data point.

Chromosomal aberration test

Root tips were grown in the absence of BrdU or thymidine and fixed one cell cycle post treatment as revealed by the differential sister-chromatid staining technique. Aberrations of the exchange type (EA) were induced exclusively. This type of aberration allows a direct comparison to SCE and chiasmata, since all 3 events require a DNA double-strand break followed by a reunion of DNA duplices. EA has been evaluated in 1000 Feulgen-stained metaphases per treatment group.

TABLE 1

RATES OF E X C H A N G E ABERRATIONS AND SISTER-CHROMATID EXCHANGES INDUCED BY UV LIGHT AND

QUINACRINE MUSTARD C O M P A R E D TO THE CHIASMA FREQUENCIES IN POLLEN MOTHER CELLS OF A STAN-

DARD LINE AND TWO O L I G O C H I A S M A T I C MUTANTS OF Vicia faba L.

Treatment Line Chiasma frequency per Aberrat ions per SCE per

pollen mother cell 1000 metaphases metaphase

(n = 100 cells a) (n = 1000 cells) (n = 75 cells)

None

UV light

s tandard 17.6 1 21.7 + 3.8

A16 9.5 0 22.4 + 3.7

A8 3.8 0 23.4 _+ 3.4

standard 17.6 91 50.6 _+ 6.8

A16 9.5 20 b 52.1 _+ 5.8

A8 3.8 9 b 46.8 + 6.3

Quinacrine standard 17.6 132 91.8 _+ 7.0

mustard AI6 9.5 46 b 91.3 _+ 6.5

A8 3.8 22 b 95.0 _+ 5.4

a As reported by Linnert and coworkers (1981).

b Significantly different compared to the values of the standard line using Student 's t-test (17< 0.025).

Statistics Student's t-test and the x2-test were performed

for comparison of SCE and EA frequencies, respectively. Differences were considered to be significant at p<0 .025 in both tests.

Results

UV light and quinacrine mustard were able to in- duce both SCE and EA. With respect to the SCE frequencies no differences between the Vicia faba lines could be stated. In the standard line as well as in the mutants A8 and A16 the background level was about 22 SCE/metaphase. After UV irradia- tion about 50 and, after quinacrine mustard treat- ment, 90 SCE/metaphase could be counted (Table 1). On the other hand, a different sensitivity in the formation of EA was found for the 3 lines observ- ed (Table 1). In cells of the standard line both mutagens induced the highest frequencies of EA, which were found in 223 metaphases out of 2000 metaphases analysed (= 100%). In the A16 mu- tant 66 out of 2000 metaphases were aberrant (=

-~ l O O

" 0 ¢-

5 0

o o o o o o o o o o o o o o o o o o o o o e o o

e o o e o o o o o o o o o o e o o o o o o o o o o o o o o o o o

o o o o o o o o 9 o o o o o e o

o o o o o o o o o o o o e o e o o o o o o e o o

, o o o o o o o o 0 o o o o o o o e

I o o o o o o

. . . . . . . i} J ° o o ° m i

:ii!il !iiiiiii

ch iasma ta

abe r ra t i ons

iiiiiiiilt STANDARD A 16 A 8

Fig. 1. Comparison of the relative frequencies of chiasmata and induced exchange aberrations in a standard line and two oligo- chiasmatic mutants of Vicia faba L.

123

30°7o) and in mutant A8 only 31 out of 2000 meta- phases (= 14%, see Fig. 1).

These findings correlate well with the reduction of visible meiotic recombination events: 17.6 chiasmata/cell (= 100%) in standard, 9.5 chias- mata/cell (= 5407o) in A16 and 3.8 chiasmata/cell (= 22070) in A8 (Fig. 1).

Moreover, the technique of differential sister- chromatid staining allows us to analyse the cell- cycle kinetics. To obtain 20°7o first-division meta- phases and 8007o differentially stained second- division metaphases the second cell cycle had to be prolonged from 12 to 18 h after UV irradiation and from 12 to 21 h after exposure to quinacrine mustard. In all 3 lines analysed the cell-cycle kinetics were the same.

Discuss ion

Crossing experiments with an oligochiasmatic mutant as one parent and a standard line of Vicia faba as the other parent had previously shown that all plants of the F1 generation are normal concern- ing their frequencies of recombination events dur- ing meiosis measured as chiasmata per pollen mother cell. The F2 generation segregated into 3 normal and 1 oligochiasmatic plants, indicating that a single recessive gene is responsible for the reduced chiasma frequency in the Vicia faba mutants.

The same phenomenon had been shown for several other oligochiasmatic plants before (see Sj6din, 1970). The oligochiasmatic lines of Vicia faba observed in the present study have been classified as asynaptic (Sj6din, 1970). Although it is extremely difficult to analyse the early stages of the meiotic prophase I, some efforts have been made to determine the kind of disturbance which leads to the reduced chiasma frequencies in the Vicia mutants. An investigation by Linnert and colleagues (1982) revealed some evidence for only partial pairing of the homologous chromosomes during pachytene stage. Our mutants, therefore, seem to be partial asynaptic.

After exposure to UV light or to quinacrine mustard the SCE frequency was increased, but not

124

d i f fe ren t , for the mu tan t s and the s t a n d a r d l ine,

while the mu tan t s were less sensi t ive to the induc-

t ion o f E A as c o m p a r e d to s t a n d a r d . These results

c on f i r m the a s s u m p t i o n tha t c h r o m o s o m a l abe r r a -

t ions and SCE are an o u t c o m e o f d i f fe ren t p ro -

cesses (Gebha r t , 1981; W o l f f , 1982).

The g o o d co r re l a t ion which had been obse rved

be tween the degree o f r educ t ion o f the ch i a sma

f o r m a t i o n and the r educ t ion o f the induced E A

f requency is in con t ras t to the f indings on somat ic

cells o f desynap t i c p lan t s (Riley and Mil ler , 1966)

and a desynap t i c h u m a n male (Pea r son et al . ,

1970). In bo th s tudies the r educed ch i a sma f o r m a -

t ion in ge rm cells was assoc ia ted with an increase

in the sensi t ivi ty to fo rm c h r o m o s o m a l a b e r r a t i o n s

in somat ic t issues exposed to X-rays . M u t a n t s are

de f ined as desynap t i c when c h i a s m a t a are not

f o r m e d despi te a n o r m a l s y n a p t o n e m a l complex

be tween the h o m o l o g o u s c h r o m o s o m e s . There-

fore , the enzyme systems ca ta lys ing r e c o m b i n a t i o n

dur ing meios is m a y be suppressed in desynap t i c

mutan t s . It has been in fe r red tha t the same en-

zymes are involved in repa i r ing D N A d a m a g e in

somat ic cells (Riley and Mil ler , 1966). This might

lead to a r educ t ion o f ch i a sma f requency as well as

to an increase o f c h r o m o s o m a l b reaks .

However , exclusively exchange- type abe r r a t i ons

occur a f te r t r e a tmen t in our Vicia faba mutan t s ,

which are supposed to be pa r t i a l a synap t i c . M o r e -

over , the rates o f abe r r a t i ons were decreased in

co r re l a t ion with the r educ t ion o f ch i a sma f requen-

cy. This f inding is in acco rdance with a s tudy

repor t ing a decreased genet ic rad iosens i t iv i ty in

meiot ic mu tan t s o f the mouse (Searle et a l . , 1969).

U n f o r t u n a t e l y , it is not m e n t i o n e d in this r epor t

whether the mice used are desynap t i c or a synap t i c

mutan t s . Since in a synap t i c ma te r i a l the enzyme

systems for D N A r e c o m b i n a t i o n are not necessar i -

ly a f fec ted , it is l ikely tha t all s tudies

d e m o n s t r a t i n g the effects o f D N A d a m a g e in

somat ic cells o f meiot ic mu tan t s a re dea l ing with

desynap t i c mutan t s . It has been m e n t i o n e d be fore

tha t the defect in mos t o f the m u t a n t s is caused by

the a l t e ra t ion o f a single gene.

The fa i lure o f the enzymes k inase or l igase m a y

lead to an increased ra te o f c h r o m o s o m a l b reaks ,

while the fa i lure o f an incis ion enzyme m a y cause

a reduced ra te o f visible c h r o m o s o m a l aber ra t ions .

There fo re , the k ind o f effects , reduced or enhanc-

ed sensi t ivi ty to D N A damage , in meiot ic mu tan t s

m a y depend on the special enzyme defect in the

ma te r i a l ana lysed . There is s t rong evidence tha t

this enzyme a l t e ra t ion affects meiot ic cells as well

as somat ic cells o f one indiv idual .

References

Friebe, B. (1979) Lack of effect of B chromosomes on sister chromatid exchanges in Secale cereale, Environ. Exp. Botany, 20, 21-26.

Gebhart, E. (1981) Sister chromatid exchanges (SCE) and struc- tural chromosome aberrations in mutagenicity testing, Hum. Genet., 58, 235-254.

Linnert, G., R. Vogel and B. Friebe (1981) Schwesterchromati- denaustausch und Chiasmabildung bei der Normalform und zwei asynaptischen Mutanten von Viciafaba L., Z. Pflanzen- ziichtg., 86, 192-200.

Natarajan, A.T., L.H.F. Mullenders, M. Meijers and U. Mukherjee (1985) Induction of sister chromatid exchanges by restriction endonucleases, Mutation Res., 144, 33-39.

Nowak, C., and G. Obe (1984) On the origin of chromosomal aberrations in human peripheral lymphocytes in vitro, I. Ex- periments with Neurospora endonuclease and polyethylene glycol, Hum. Genet., 66, 335-343.

Obe, G., F. Palitti, C. Tanzarella, F. Degrassi and R. De Salvia (1985) Chromosomal aberrations induced by restriction endonucleases, Mutation Res., 150, 359-368.

Pearson, P.L., J.D. Ellis and H.J. Evans (1970) A gross reduc- tion in chiasma formation during meiotic prophase and a defective DNA repair mechanism associated with a case of human male infertility, Cytogenetics, 9, 460-467.

Riley, R., and T.E. Miller (1966) The differential sensitivity of desynaptic and normal genotypes of barley to X-rays, Muta- tion Res., 3, 355-359.

Searle, A.G., R.J. Berry and C.V. Beechey (1970) Cytogenetic radiosensitivity and chiasma frequency in wild-living male mice, Mutation Res., 9, 137-140.

Sj6din, J. (1970) Induced asynaptic mutants in Viciafaba L., Hereditas, 66, 215-232.

Wolff, S. (1982) Sister Chromatid Exchange, Wiley, New York, pp. 41-57.

Communicated by H. Greim