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The production of monosomic-trisomic individuals in Drosophila melanogaster by X-irradiation of immature oocytes
429
SHORT COMMUNICATIONS
18 SWANN, P. F., AND P. N. MAGEE, N i t r o s a n l i n e - i n d u c e d carcinogenesis, Biochem. J., io (1968) 39-47. 19 TOTH, B., P. N. MAGEE AND P. SHUBIK, Carcinogenesis s t u d y w i t h d i m e t h y l n i t r o s a m i n e a d n l i n i s t e r e d orally to a d u l t a n d s u b c u t a n e o u s l y to n e w b o r n B A L B / c mice, Cancer Res., 24 (1964) 1712-1716. 20 UDENFRIEND, S., C. T. CLARK, J. AXELROD AND B. 13. BRODIE, Ascorbic acid in a r o m a t i c h y d r o x y l a t i o n , 1. A m o d e l s y s t e m for a r o m a t i c h y d r o x y l a t i o n , J. Biol. Chem., 208 (I954) 731-739 .
Received June 7th, 1971 Revision received September 6th, 1971 Mutation Res., 13 (I97I) 425 429 Mutation Research Elsevier P u b l i s h i n g Conlpany, A m s t e r d a m P r i n t e d in T h e N e t h e r l a n d s
The production of monosomic-trisomic individuals in
Drosophila
melanogaster by X-irradiation of immature oocytes It has been shown recently ~ that a certain fraction of the X-chromosome losses induced by X-rays in immature oocytes of Drosophila melanogaster are partial ones, i.e. losses not of the whole but of only part of the X-chromosome. This fraction, however, could not be determined exactly in that work because some uncertainty existed concerning the classification of a special type of X-loss individuals. These flies carried a dot-like chromosome (chromosomal fragment ?), called "point" in that paper 4, of the same size as chromosome IV. We were not sure whether such "points" were indeed chromosomes IV or small X-chromosomal fragments. The first interpretation would have presupposed a very high frequency of triplo-IV individuals (produced by maternal nondisjunction) among the XO flies. The second explanation would have been in line with our finding that dot-like sex-chromosomal fragments of about the same and in some cases exactly the same size as chromosome IV, represent a relatively large proportion of sex-chromosome losses (complete plus partial) induced by X-rays in mature spermatozoa of Drosophila melanogaster5. Leaving open to discussion the question of the nature of the "points", we stated 4 that the proportion of partial X losses induced by X-rays (3500 R) in immature oocytes would be either 7% (3/43) if the "points" were chromosomes IV or 230//0 (lO/43) if they were of X-chromosomal origin. The difference between the numerators IO--3--~7 represents the number of unclassified cases among the 43 X-loss larvae 4. Both in order to decide which of these two explanations is the right one and to determine the proportion of partial losses more exactly than in the former study 4, the following experiments were carried out. Cerebral ganglia of F1 larvae originating from complete or partial X loss induced by X-rays in immature oocytes of wild-type females were analysed. However, deviating from the earlier work, the metaphase chromosomes were first stained with the fluorochrome-quinacrine dihydrochloride ("Atebrin", G. Gurr, Ltd.). In order to check the quinacrine results, the chromosomes were stained with orcein after the fluorescence microscopical analysis. As has been demonstrated recently 6, the fourth chromosome is stronger fluorescence-wise Mutation Res., 13 (1971) 429-432
43 °
SHORT COMMUNICATIONS
TABLE I T H E P O S I T I V E C O R R E L A T I O N B E T \ V E E N T H E I N D U C T I O N OF X - C H R O M O S O M E IV
NOND1SJUNCTION
AFTER
X-IRRADIATION
OF I M M A T U R E
O O C Y T E S OF
LOSS A N D C H R O M O S O M E
D1/oso/)hila t}lFla~lomoa,stt'Y
WITH 3500 R
.4 berralion
Number oj cases belonging to (-)rcein experiments a Quirmcrine e.rperiments a
Rod-X fragment Rmg-X fragnIent X(). triplo-lV Cornplete X loss
3b 7 3,3I,
3 I 7 3t b
Total
4.3
42
a See text for explanation. 1, ()he exanlple with one chromosome IV only. t h a n a n y other cllromosome of D r o s o p h i l a melanogaster, except for p a r t s of tile Y. A l t h o u g h a short region at the centromere of tile X - c h r o m o s o m e is r e l a t i v e l y intensely fluorescent too, a distinction between t h a t region a n d the IV can be m a d e because of the brighter fluorescence of the l a t t e r chronlosome. This distinction was necessary because the " p o i n t s " would consist m a i n l y of the region near to tile cent r o m e r e of the X if t h e y were X - c h r o m o s o m a l fragments. N o r m a l females (Berlin wild) were i r r a d i a t e d with 3500 R X - r a y s and then l n a t e d to y males. F1 individuals originating from t o t a l or p a r t i a l loss of the m a t e r n a l X were y a n d could be recognized as e a r l y as the l a r v a l stage b y their yellowish m o u t h p a r t s . Only stage- 7 oocytes were analysed. The details of the biological a n d i r r a d i a t i o n conditions of our e x p e r i m e n t s were the same as described in ref. 4. The quinacrine staining was p e r f o r m e d as in the work of VOSA6, except t h a t the p r e p a r a tions were s t a i n e d for 3 ° rain i n s t e a d of for 3-5 rain. After the fluorescence micros c o p i c a l a n a l y s i s , the cover slips were r e m o v e d b y inunersing the slides in absolute alcohol. Then the p r e p a r a t i o n s were stained with orcein in the same w a y as in our former e x p e r i m e n t s 4,~. Table I shows our results ("quinacrine e x p e r i m e n t s " ) together with those obt a i n e d earlier 4 ("orcein e x p e r i m e n t s " ) under the same biological and physical conditions. The a g r e e m e n t between b o t h series is r e m a r k a b l y good. The fluorescence microscopical analysis of the seven cases which were c h a r a c t e r i z e d b y a t h i r d " p o i n t " d e m o n s t r a t e d t h a t each of t h e m was a t r i p l o - I V individual, the " p o i n t " being a fourth chromosome. Fig. I represents three of those seven cases, i.e. individuals of the m o n o - X , t r i p l o - i V constitution. Because the v i a b i l i t y of n o n d i s j u n c t i o n a l haploIV individuals is reduced, a c o r r e s p o n d i n g l y high frequency of m o n o - X , h a p l o - I V flies was not found. The few cases observed are listed in Table I. I t seems l e g i t i m a t e to assume t h a t also the seven cases with a t h i r d " p o i n t " observed in the earlier work ~ were mono-X, t r i p l o - I V individuals (see "orcein e x p e r i m e n t s " of Table I). The result of the orcein staining which followed the fluorescence analysis in the " q u i n a c r i n e e x p e r i m e n t s " did not reveal a n y inconsistencies, although the n a t u r e of the " p o i n t s " would not have been clarified b e y o n d d o u b t by orcein staining alone. In m a r k e d c o n t r a s t to this finding, the " p o i n t s " observed after X - i r r a d i a t i o n of m a t u r e s p e r m a tozoa 5 were small sex-chronmsomat fragments. Their s o m e w h a t greater size variability, as c o m p a r e d to the more c o n s t a n t size of the " p o i n t s " found after i r r a d i a t i o n of 5lutation Res., t 3 (~97t) 420 4132
SHORT COMMUNICATIONS
72'
431
:7
o
e
Fig. I. Fluorescence microscopical d e m o n s t r a t i o n (fluorochromc q u i n a c r i n e dihydrochloride) ot t h e p r o d u c t i o n of XO, triplo-IV individuals b y X - i r r a d i a t i o n of i m m a t u r e oocytes of Drosophila melauogaster (3 cases). M e t a p h a s e s of larval cerebral ganglia were a n a l y s e d . T h e b r i g h t l y fluorescent dot-like c h r o m o s o m e s IV can be clearly seen. Note also t h e relatively s t r o n g fluorescence of t h e regions near to t h e c e n t r o m e r e of t h e X a n d of t h e third c h r o m o s o m e s .
immature oocytes ~, agrees with this statement. (In both series tile analysis was performed after orcein staining.) The frequency of 3' larvae originating from complete and partial X loss, based on the total number of larvae analysed, was 5.3°o (99/1857). This value agrees well with that obtained in the former experiments 4, namely 4.7°/'0. Firstly, our results allow the calculation of the proportion of partial X losses induced by X-rays (350o R) in immature oocytes of Drosophila melmwgaster more exactly than in the earlier work ~. These proportions are 9.5% (4/42) in the quinacrine and 7.o% (3/43) in the orcein experiments (see Table I), the combined frequency O/ O/ being 8.2/o (7/85) with 95°,o confidence limits of 3.4 and 16.2/o, respectively. Secondly, our results demonstrate a relatively high frequency of triplo-IV individuals among XO flies (complete X loss), namely 18.4% (7/38) in the quinacrine ~'~Iutalio~ Res., 13 (1971) 420-432
432
SHORT COMMUNICATIONS
and I7.5°o (7/40) in the oreein experiments (see Table I). The combined frequency is 17.9% (14/78) with 95~o confidence limits of lO.2 and 28.3%, respectively. Although there exist no data concerning the frequency of chromosome IV nondisjunction (not correlated with X loss) induced by X-rays in immature oocytes of females with free X-chromosomes, it can be estinlated from indirect evidencea, s that after irradiation with 35oo R this frequency is less than 1% (when calculated in such a way that it can be compared with the frequency of I7.9°/o mentioned above). One difficulty in deriving this frequency more exactly is that it should be based exclusively on offspring with normal sex-chromosonle constitution (XX and XY), in opposition to the value of i7.9°~ based on XO individuals only. Nevertheless, there is no doubt that the fiequency of triplo-IV individuals is much higher when estimated with respect to XO males instead of X X or XY flies. This demonstrates a positive correlation between the
X-ray induction of complete X-chromosome loss and chromosome I V nondisjunctio~z in immature oocvtes of Dros@hila melanogaster. We do not know, however, whether the X losses correlated with nondisjunction of chromosomes 1V result from Xchronlosonml nondisjunction or from "true" X losses (via breakage of the X). The "inverse question", namely what fraction of the triplo-IV individuals is correlated with complete X loss, cannot be answered satisfactorily, because the frequency of chromosome IV nondisjunction induced by 35oo R in inunature oocytes of flies with normal sex-chromosonle constitution is not known exactly. A possible mechanism underlying the correlation observed might be radiationinduced interchange between X and IV, followed by the separation of the heterologues at the first meiotic division. Consequently, the homologues of the interchangeinvolved X and IV might segregate more or less at random, thus producing (among other nondisjunctional types) nullo-X, diplo-IV gametes. This attractive hypothesis has been derived by PARI
Institute for Radiation Biology, U~dversitv of M#nster, Miinster (Germa~oJ)
H. TRAUT W. SCHFAI)
[ GRELL, R. l;., IL. 11. ~IUI~OZ ANt) X*V. F. KIRSCHBAUI~I,Radiat i o n - i n d u c e d non-disjunction a n d loss of chromosomes in Drosophila melanogaster females, 1 .The effect of chromosome size, Mutation Rrs., 3 (1966) 494-5o2. 2 PARKER, D. R., Coordinate nondisjunction of Y and fourth chroinosomes in irradiated c o m p o u n d X female Drosophila, Mutation Res., 9 (197o) 307 322. .] TRAUT, H., The dose-dependence of X - c h r o m o s o m e loss and nondisjunction induced by X rays in oocytes of Drosophila melanogaster, Mutation i~es., t (I964) 157 162. 4 "FRAUT, H., AND W. ~CHEID, Cytological analysis of partial and total X - c h r o m o s o m e loss induced by X - r a y s in oocytes of Drosophila melanogaster, Mutation Res., io (197 o) 583-589 . 5 TRAUT, H., W. ~CHEll) AN[) H . W1ND, P a r t i a l a n d t o t a l s e x - c h r o m o s o m e loss i n d u c e d by X - r a y s in m a t u r e s p e r m a t o z o a of Drosophila melanogaster, Mutation Res., 9 (t97 o) 489 499. 6 VOSA, C. G., The discriminating fluorescence p a t t e r n s of the chromosomes of Drosophila mclanogash'r, Ckromosonza, 31 (197o) 446-451.
Received August 23rd, 1971 ~llutation l?es., 13 (I971) 429-432
SHORT COMMUNICATIONS
18 SWANN, P. F., AND P. N. MAGEE, N i t r o s a n l i n e - i n d u c e d carcinogenesis, Biochem. J., io (1968) 39-47. 19 TOTH, B., P. N. MAGEE AND P. SHUBIK, Carcinogenesis s t u d y w i t h d i m e t h y l n i t r o s a m i n e a d n l i n i s t e r e d orally to a d u l t a n d s u b c u t a n e o u s l y to n e w b o r n B A L B / c mice, Cancer Res., 24 (1964) 1712-1716. 20 UDENFRIEND, S., C. T. CLARK, J. AXELROD AND B. 13. BRODIE, Ascorbic acid in a r o m a t i c h y d r o x y l a t i o n , 1. A m o d e l s y s t e m for a r o m a t i c h y d r o x y l a t i o n , J. Biol. Chem., 208 (I954) 731-739 .
Received June 7th, 1971 Revision received September 6th, 1971 Mutation Res., 13 (I97I) 425 429 Mutation Research Elsevier P u b l i s h i n g Conlpany, A m s t e r d a m P r i n t e d in T h e N e t h e r l a n d s
The production of monosomic-trisomic individuals in
Drosophila
melanogaster by X-irradiation of immature oocytes It has been shown recently ~ that a certain fraction of the X-chromosome losses induced by X-rays in immature oocytes of Drosophila melanogaster are partial ones, i.e. losses not of the whole but of only part of the X-chromosome. This fraction, however, could not be determined exactly in that work because some uncertainty existed concerning the classification of a special type of X-loss individuals. These flies carried a dot-like chromosome (chromosomal fragment ?), called "point" in that paper 4, of the same size as chromosome IV. We were not sure whether such "points" were indeed chromosomes IV or small X-chromosomal fragments. The first interpretation would have presupposed a very high frequency of triplo-IV individuals (produced by maternal nondisjunction) among the XO flies. The second explanation would have been in line with our finding that dot-like sex-chromosomal fragments of about the same and in some cases exactly the same size as chromosome IV, represent a relatively large proportion of sex-chromosome losses (complete plus partial) induced by X-rays in mature spermatozoa of Drosophila melanogaster5. Leaving open to discussion the question of the nature of the "points", we stated 4 that the proportion of partial X losses induced by X-rays (3500 R) in immature oocytes would be either 7% (3/43) if the "points" were chromosomes IV or 230//0 (lO/43) if they were of X-chromosomal origin. The difference between the numerators IO--3--~7 represents the number of unclassified cases among the 43 X-loss larvae 4. Both in order to decide which of these two explanations is the right one and to determine the proportion of partial losses more exactly than in the former study 4, the following experiments were carried out. Cerebral ganglia of F1 larvae originating from complete or partial X loss induced by X-rays in immature oocytes of wild-type females were analysed. However, deviating from the earlier work, the metaphase chromosomes were first stained with the fluorochrome-quinacrine dihydrochloride ("Atebrin", G. Gurr, Ltd.). In order to check the quinacrine results, the chromosomes were stained with orcein after the fluorescence microscopical analysis. As has been demonstrated recently 6, the fourth chromosome is stronger fluorescence-wise Mutation Res., 13 (1971) 429-432
43 °
SHORT COMMUNICATIONS
TABLE I T H E P O S I T I V E C O R R E L A T I O N B E T \ V E E N T H E I N D U C T I O N OF X - C H R O M O S O M E IV
NOND1SJUNCTION
AFTER
X-IRRADIATION
OF I M M A T U R E
O O C Y T E S OF
LOSS A N D C H R O M O S O M E
D1/oso/)hila t}lFla~lomoa,stt'Y
WITH 3500 R
.4 berralion
Number oj cases belonging to (-)rcein experiments a Quirmcrine e.rperiments a
Rod-X fragment Rmg-X fragnIent X(). triplo-lV Cornplete X loss
3b 7 3,3I,
3 I 7 3t b
Total
4.3
42
a See text for explanation. 1, ()he exanlple with one chromosome IV only. t h a n a n y other cllromosome of D r o s o p h i l a melanogaster, except for p a r t s of tile Y. A l t h o u g h a short region at the centromere of tile X - c h r o m o s o m e is r e l a t i v e l y intensely fluorescent too, a distinction between t h a t region a n d the IV can be m a d e because of the brighter fluorescence of the l a t t e r chronlosome. This distinction was necessary because the " p o i n t s " would consist m a i n l y of the region near to tile cent r o m e r e of the X if t h e y were X - c h r o m o s o m a l fragments. N o r m a l females (Berlin wild) were i r r a d i a t e d with 3500 R X - r a y s and then l n a t e d to y males. F1 individuals originating from t o t a l or p a r t i a l loss of the m a t e r n a l X were y a n d could be recognized as e a r l y as the l a r v a l stage b y their yellowish m o u t h p a r t s . Only stage- 7 oocytes were analysed. The details of the biological a n d i r r a d i a t i o n conditions of our e x p e r i m e n t s were the same as described in ref. 4. The quinacrine staining was p e r f o r m e d as in the work of VOSA6, except t h a t the p r e p a r a tions were s t a i n e d for 3 ° rain i n s t e a d of for 3-5 rain. After the fluorescence micros c o p i c a l a n a l y s i s , the cover slips were r e m o v e d b y inunersing the slides in absolute alcohol. Then the p r e p a r a t i o n s were stained with orcein in the same w a y as in our former e x p e r i m e n t s 4,~. Table I shows our results ("quinacrine e x p e r i m e n t s " ) together with those obt a i n e d earlier 4 ("orcein e x p e r i m e n t s " ) under the same biological and physical conditions. The a g r e e m e n t between b o t h series is r e m a r k a b l y good. The fluorescence microscopical analysis of the seven cases which were c h a r a c t e r i z e d b y a t h i r d " p o i n t " d e m o n s t r a t e d t h a t each of t h e m was a t r i p l o - I V individual, the " p o i n t " being a fourth chromosome. Fig. I represents three of those seven cases, i.e. individuals of the m o n o - X , t r i p l o - i V constitution. Because the v i a b i l i t y of n o n d i s j u n c t i o n a l haploIV individuals is reduced, a c o r r e s p o n d i n g l y high frequency of m o n o - X , h a p l o - I V flies was not found. The few cases observed are listed in Table I. I t seems l e g i t i m a t e to assume t h a t also the seven cases with a t h i r d " p o i n t " observed in the earlier work ~ were mono-X, t r i p l o - I V individuals (see "orcein e x p e r i m e n t s " of Table I). The result of the orcein staining which followed the fluorescence analysis in the " q u i n a c r i n e e x p e r i m e n t s " did not reveal a n y inconsistencies, although the n a t u r e of the " p o i n t s " would not have been clarified b e y o n d d o u b t by orcein staining alone. In m a r k e d c o n t r a s t to this finding, the " p o i n t s " observed after X - i r r a d i a t i o n of m a t u r e s p e r m a tozoa 5 were small sex-chronmsomat fragments. Their s o m e w h a t greater size variability, as c o m p a r e d to the more c o n s t a n t size of the " p o i n t s " found after i r r a d i a t i o n of 5lutation Res., t 3 (~97t) 420 4132
SHORT COMMUNICATIONS
72'
431
:7
o
e
Fig. I. Fluorescence microscopical d e m o n s t r a t i o n (fluorochromc q u i n a c r i n e dihydrochloride) ot t h e p r o d u c t i o n of XO, triplo-IV individuals b y X - i r r a d i a t i o n of i m m a t u r e oocytes of Drosophila melauogaster (3 cases). M e t a p h a s e s of larval cerebral ganglia were a n a l y s e d . T h e b r i g h t l y fluorescent dot-like c h r o m o s o m e s IV can be clearly seen. Note also t h e relatively s t r o n g fluorescence of t h e regions near to t h e c e n t r o m e r e of t h e X a n d of t h e third c h r o m o s o m e s .
immature oocytes ~, agrees with this statement. (In both series tile analysis was performed after orcein staining.) The frequency of 3' larvae originating from complete and partial X loss, based on the total number of larvae analysed, was 5.3°o (99/1857). This value agrees well with that obtained in the former experiments 4, namely 4.7°/'0. Firstly, our results allow the calculation of the proportion of partial X losses induced by X-rays (350o R) in immature oocytes of Drosophila melmwgaster more exactly than in the earlier work ~. These proportions are 9.5% (4/42) in the quinacrine and 7.o% (3/43) in the orcein experiments (see Table I), the combined frequency O/ O/ being 8.2/o (7/85) with 95°,o confidence limits of 3.4 and 16.2/o, respectively. Secondly, our results demonstrate a relatively high frequency of triplo-IV individuals among XO flies (complete X loss), namely 18.4% (7/38) in the quinacrine ~'~Iutalio~ Res., 13 (1971) 420-432
432
SHORT COMMUNICATIONS
and I7.5°o (7/40) in the oreein experiments (see Table I). The combined frequency is 17.9% (14/78) with 95~o confidence limits of lO.2 and 28.3%, respectively. Although there exist no data concerning the frequency of chromosome IV nondisjunction (not correlated with X loss) induced by X-rays in immature oocytes of females with free X-chromosomes, it can be estinlated from indirect evidencea, s that after irradiation with 35oo R this frequency is less than 1% (when calculated in such a way that it can be compared with the frequency of I7.9°/o mentioned above). One difficulty in deriving this frequency more exactly is that it should be based exclusively on offspring with normal sex-chromosonle constitution (XX and XY), in opposition to the value of i7.9°~ based on XO individuals only. Nevertheless, there is no doubt that the fiequency of triplo-IV individuals is much higher when estimated with respect to XO males instead of X X or XY flies. This demonstrates a positive correlation between the
X-ray induction of complete X-chromosome loss and chromosome I V nondisjunctio~z in immature oocvtes of Dros@hila melanogaster. We do not know, however, whether the X losses correlated with nondisjunction of chromosomes 1V result from Xchronlosonml nondisjunction or from "true" X losses (via breakage of the X). The "inverse question", namely what fraction of the triplo-IV individuals is correlated with complete X loss, cannot be answered satisfactorily, because the frequency of chromosome IV nondisjunction induced by 35oo R in inunature oocytes of flies with normal sex-chromosonle constitution is not known exactly. A possible mechanism underlying the correlation observed might be radiationinduced interchange between X and IV, followed by the separation of the heterologues at the first meiotic division. Consequently, the homologues of the interchangeinvolved X and IV might segregate more or less at random, thus producing (among other nondisjunctional types) nullo-X, diplo-IV gametes. This attractive hypothesis has been derived by PARI
Institute for Radiation Biology, U~dversitv of M#nster, Miinster (Germa~oJ)
H. TRAUT W. SCHFAI)
[ GRELL, R. l;., IL. 11. ~IUI~OZ ANt) X*V. F. KIRSCHBAUI~I,Radiat i o n - i n d u c e d non-disjunction a n d loss of chromosomes in Drosophila melanogaster females, 1 .The effect of chromosome size, Mutation Rrs., 3 (1966) 494-5o2. 2 PARKER, D. R., Coordinate nondisjunction of Y and fourth chroinosomes in irradiated c o m p o u n d X female Drosophila, Mutation Res., 9 (197o) 307 322. .] TRAUT, H., The dose-dependence of X - c h r o m o s o m e loss and nondisjunction induced by X rays in oocytes of Drosophila melanogaster, Mutation i~es., t (I964) 157 162. 4 "FRAUT, H., AND W. ~CHEID, Cytological analysis of partial and total X - c h r o m o s o m e loss induced by X - r a y s in oocytes of Drosophila melanogaster, Mutation Res., io (197 o) 583-589 . 5 TRAUT, H., W. ~CHEll) AN[) H . W1ND, P a r t i a l a n d t o t a l s e x - c h r o m o s o m e loss i n d u c e d by X - r a y s in m a t u r e s p e r m a t o z o a of Drosophila melanogaster, Mutation Res., 9 (t97 o) 489 499. 6 VOSA, C. G., The discriminating fluorescence p a t t e r n s of the chromosomes of Drosophila mclanogash'r, Ckromosonza, 31 (197o) 446-451.
Received August 23rd, 1971 ~llutation l?es., 13 (I971) 429-432