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Effects of 2,4-dinitrophenol and dinactin on heat-sensitive and ecdysone-specific puffs of Drosophila salivary gland chromosomes in vitro
Cell Differentiation 2, 221-228 (1973). © North-Holland Publishing Company
EFFECTS OF 2,4-DINITROPHENOL
AND DINACTIN ON
HEAT-SENSITIVE AND ECDYSONE-SPECIFIC PUFFS OF
DROSOPHILA
SALIVARY GLAND CHROMOSOMES
IN V I T R O
Ludger RENSING L Zoologisches lnstitut der Universitilt Ggttingen, GOttingen, Western Germany Accepted 10 June 1973
Salivary glands from third instar larvae of Drosophila melanogaster were incubated in vitro with various concentrations of 2,4-dinitrophenol or dinactin and the effects on puff size were measured. At lower concentrations of either substance (10-a M DNP and 10.7 M dinactin) the sizes of the heat-sensitive series of puffs are significantly enlarged. At higher concentrations (10-~ M and 10-4 M) ecdysone-spccific puffs and puffs that appear somewhat later in the course of development are induced whereas the heat-sensitive puffs do not react. A regression of puff size can be observed at higher concentrations in the case of the puffs at 63E and 47BC. The induction of the heat-sensitive series of puffs by dinactin is development-specific: the reactivity of these puffs is maximal at a developmental age of about 117 hr after egg laying (25°C). The normal release of ecdysone during development at about 115 hr as well as the incubation with 1 ~tg/ml or 10/~g/ml ecdysone results in a reduced reactivity to dinactin of puff 87B in particular. A similar effect can be obtained when 10-5 M ouabain is applied.
A certain series of puffs in the salivary glands of Drosophila melanogaster has been shown to be inducible by heat, uncoupling substances (Ritossa, 1963, 1964; Ashburner, 1970; Ellgaard, 1972), and by puromycin and fusidic acid (Schoon et al., 1973). The inductive mechanism may include certain proteins as can be concluded from the latter experiments and the results of Helmsing et al. (1971); it may also include changes in the respiratory metabolism (Leenders et al., 1972a,b). Most substances that uncouple oxidative phosphorylation and induce a specific group of puffs, as for example 2,4-dinitrophenol (DNP), dicumarol, salicylate etc., affect membrane permeabilities for cations, ATPase activity, ATP level and respiration. Which of these effects are of primary importance in the inductive mechanism is far from clear; the same is true for the role of K+-concentrations, cellular and nuclear membrane potentials and their alterations by hormones and cyclic adenosine monophosphate (Kroeger, 1966; Leenders et al., 1970; Lezzi et al., 1972; Rensing et al., 1972). In order to analyse the effects of uncoupling substances in more detail, DNP and an uncoupling antibiotic, dinactin, were tested over a wide range of concentra-
222
Experimental changes in pufft'ng activity in Drosophila
tions. The inductive effect of the antibiotic was registered also under different developmental conditions and under the influence o f ecdysone and ouabain.
MATERIALS AND METHODS Larvae of an inbred stock of D. melanogaster were reared at 25°C under a light-dark cycle of 12:12 hr. At the end of the third instar, the salivary glands were excised, sister glands separated and kept in an artificial medium (modified after Rensing, 1969; Nagel, in preparation). In most cases one of the sister glands was incubated with the substance or antibiotic for 2 hr whereas the other gland served as a control. Squashing of the salivary glands and measurements of the puff size were the same as in experiments described earlier (Rensing et al., 1972): the width of the puff divided by the width of a neighbouring band (puff ratio) of experimental and control glands was taken and the mean and threefold standard error calculated. The following substances were used; 2,4-dinitrophenol (Merck, Darmstadt), dinactin (a gift from Ciba/Geigy, Basel), ouabain (g-strophantin, Merck, Darmstadt), and ecdysone (a gift from Schering, Berlin).
RESULTS Incubation of a sister salivary gland with different concentrations of DNP led to the following results when compared with the other untreated sister gland (fig. 1).
+
15 ~
]25
/~ ,...~, 63Bc
lo-
+ ~i 878
+
"
478C
~
63E
-4 -3 -2 (-1) M Fig. h Effect. of 2,4 dinitrophenol on puff size in salivary gland chromosomes after 2 hr of incubation (third instar larvae, -117 hr). Ordinate: puff ratio of the treated sister gland divided by the puff ratio of the untreated control gland; abscissa: molar concentrations of DNP, (-1) indicates a saturated solution.
L. RENSING
223
j
,
74EF
•
71C
,
1.25 5oCD
1.o
-4
-3
-2
(-I) ~
Fig. 2. Effect of 2,4-dinitrophenol on puff size in salivary gland chromosomes; details as in fig. 1.
Heat-sensitive puffs (Ashburner, 1970), as represented by puffs 63BC and 87B, show maximal increase in puff size at 10-3 M, less increases at 10-4 M, 10-~ M and 10-a M in the case of 63BC and no increase at all at 10-4 M and 10-1 M in the case of 87B. These findings were in line with the earlier observations of Ritossa (1964). A regression of puff size after the incubation period can be noted in 47BC and 63E at concentrations higher than 10-~ M. Development-specific puffs, as represented by puffs that react directly on the release of ecdysone (74EF, 2B, and possibly 50CD) and by puffs activated some hours later in the course of development (71C and 85F), increase their size after incubation with 10-3-10 -1 M DNP significantly (fig. 2). Since the latter effects were observed at very high concentrations and even with a saturated solution of DNP, conditions that bring'about unphysiologic osmotic values, similar experiments with an uncoupling antibiotic, dinactin, were performed. In these experiments the heat-sensitive group of puffs is significantly enlarged at concentrations of 10-9-10-s M dinactin (fig. 3). Maximum size can be observed at 10-7 M in the case of 87B and 03D and at 10-6 M in the case of 63BC. A regression of puffs 47BC and 63E occurs at high concentrations (10 -4 M). The sizes of ecdysone-specific puffs reach a peak at 10-s M (50CD) and 10-4 M (74EF and 2B) whereas later appearing puffs are maximally enlarged at 10-3 M (71C and 85F) as shown in fig. 4. These enlargements must be due to an actual increase in size rather than to an inhibition of the regression process because the control glands do not show any of these development-specific puffs. If these puffs are present at the time when the glands are excised, they are preserved during the incubation in the normal medium. In an attempt to analyse the mechanisms involved in the induced changes of the puffing pattern, it seemed of interest to test the reactivity of the heat-sensitive loci in the course of development. The developmental stage of third instar larvae was
Experimental changes in puffing activity in Drosophila
224
1.75
1.50
1.25
1.o 87B 93D 63E
0:75
-9
-8
-7
-6
-5
-4
-3
M
Fig. 3. Effect of dinactin on p u f f size o f salivary gland c h r o m o s o m e s after 2 hr of incubation; details as in fig. l, abscissa: molar concentrations of dinactin.
74 EF 1.5o
v1? :/.
125
I
2B
71C 85F
5oCD 1.o
-9
-8
-7
-6
-5
-4
-3
M
Fig. 4. Effect o f dinactin on p u f f size in salivary gland c h r o m o s o m e s ; details as in fig. l. abscissa: molar concentrations o f dinactin.
L. RENSING
225
determined following criteria derived from the behaviour of the larvae and the morphology of the salivary glands (Rensing et al., 1972) and also from the puffing pattern (Ashburner, 1967). At the age of 115 hr, characterized by the appearance of ecdysone-specific puffs, the reactivity of heat-sensitive puffs is low when incubated with 10-7 M and 10-6 M dinactin (fig. 5). A maximum reactivity is observed at a time between the release of ecdysone and puparium formation; immediately before puparium formation (120 hr) the reactivity decreases again. If the lower reactivity of the heat-sensitive group of puffs at the developmental age of 115 hr could be related to the release of ecdysone and the induction of ecdysone-specific puffs at that time, addition of ecdysone to the dinactin containing medium should result in a similarly reduced reactivity to the uncoupling substance. This is actually observed in a series of experiments using 1 #g/ml or 10 ~g/ ml ecdysone (table 1), especially significant in the case of puff 87B. The inductive effect of ecdysone, on the other hand, is evident from the significant increase in the size of puff 74EF. No changes at all can be measured in puffs 50CD and 47BC, findings that confirm the validity of the method. Similar results as to the reduced reactivity to dinactin of some puffs are obtained by incubating the salivary glands with the steroid rhamnoside, ouabain (table 1). Ouabain specifically inhibits the activity of the Na ÷, K÷-dependent ATPase. After a
175t
o ~./z°, ~. 1C~o1 °'//'~" ~÷ 125t lo
075
/"
115
87B 63BC 63E 117
'12oh
Fig. 5. Effect of dinactin on puff size in salivary gland chromosomes at different times during development. Ordinate: as in fig. 1, abscissa: developmental age of third instar larvae (Ashburner, 1967). o - - o 10 -7 M, + - - + 10 -6 M dinactin.
226
Experimental changes in puffing activity in Drosophila
Table 1 Effect of the combined incubation (2 hr) with 10-6 M dinactin and 10-5 M ouabain and with 10-7 M dinactin and 1 or 10 ~tg/ml ecdysone as compared to the effect of dinactin alone. Values represent the puff ratios together with the threefold standard error. Puff sites:
63BC
63E
87B
93D
Controls 10-6 M dinactin
1.74_+ 0.13
1.22-+ 0.12
2.02-+ 0.18
2.53_+ 0.36
10-6 M dinactin + 10-5 M ouabain
1.55 _+0.09
1.32 -+ 0.18
1.43 -+ 0.14
2.64 _+0.31
Controls 10-TM dinactin
1.39_+ 0.10
1.18-+ 0.09
1.84_+ 0.18
2.62-+ 0.6
1.30 -+ 0.06
I. 13 -+ 0.06
1.41 -+ 0.16
2.43 _+ 0.5
Puff sites:
47B
50C1)
74EF
2B
Controls 10-6 M dinactin
1.80-+ 0.2
1.69-+ 0.34
1.58-+ 0.24
1.36-+ 0.11
10-6 M dinactin + 10-5 M ouabain
1.70 -+ 0.2
1.70 -+ 0.15
1.64 -+ 0.29
1.40 -+ 0.08
Controls 10-7 M dinactin
1.54 -+ 0.2
1.56 -+ 0.15
1.31 -+ 0.2
1.43 -+ 0.25
10-7 M dinactin + ecdysone (1 or 10 ~g/ml)
1.52-+ 0.13
1.52-+ 0.21
1.73-+ 0.09
1.44 + - 0.18
10 -7 M dinactin
+ ecdysone (1 or 10 ~g/ml)
c o m b i n e d t r e a t m e n t o f t h e glands w i t h b o t h s u b s t a n c e s ( 1 0 -s M o u a b a i n , 10 -6 M d i n a c t i n ) t h e sizes o f puffs 6 3 B C a n d 8 7 B are significantly r e d u c e d as c o m p a r e d w i t h t h o s e glands i n c u b a t e d w i t h d i n a c t i n alone.
DISCUSSION T h e h e a t - s e n s i t i v e series o f puffs ( A s h b u r n e r , 1 9 7 0 ) has b e e n i n d u c e d b y a n u m b e r o f c h e m i c a l u n c o u p l e r s o f o x i d a t i v e p h o s p h o r y l a t i o n , such as 3 × l 0 -3 M s o d i u m azide, 10 -~ M s o d i u m salicylate, 10 -3 M d i c u m a r o l a n d 10 -3 M D N P (Ritossa, 1 9 6 4 ) . T o this list o f s u b s t a n c e s u n c o u p l i n g a n t i b i o t i c s can be a d d e d : 10 - 9 10 -6 M d i n a c t i n , as d e m o n s t r a t e d in this p a p e r , 1 0 - T M m o n a c t i n or t r i n a c t i n (Behnel, u n p u b l . ) a n d 2 × 10 -s M v a l i n o m y c i n ( S t e f f e n et al., u n p u b l . ) w h i c h all i n d u c e t h e same series o f puffs. T h e u n c o u p l i n g m e c h a n i s m a n d o t h e r related effects m a y be similar in t h e case o f u n c o u p l i n g a n t i b i o t i c s a n d c h e m i c a l u n c o u p l e r s : a) a n increase in t h e selective p e r m e a b i l i t y o f m e m b r a n e s for c a t i o n s , especially for K ÷ as it is k n o w n for t h e n o n a c t i n a n t i b i o t i c s ( K i l b o u r n et al., 1 9 6 7 ; Eigen et al., 1970), for v a l i n o m y c i n ( M o o r e et al., 1 9 6 4 ; L~iuger, 1 9 7 2 ) , a n d salicylate ( L e v i t a n et al.,
L. RENSING
227
1972); b) an increase in ATPase activity as demonstrated in the case of DNP and valinomycin (McMurray, 1959), and the nonactin antibiotics (Graven et al., 1966); c) a stimulation of respiration, as for example measured after treatment with DNP (Slater, 1963), nonactin antibiotics (Graven et al., 1966), and valinomycin (Ogata et al., 1966). Some of these effects can also be obtained by temperature changes and reversal of anaerobiosis, conditions that at the same time induce the series of puffs, that respond to the uncoupling substances (reviews, see Berendes, 1972; Ashburner, 1972). Inductions of ecdysone-specific and juvenile hormone-specific puffs have been related to changes of the Na÷/K÷-ratio within the nucleus (Kroeger, 1966; Robert, 1971; Lezzi et al., 1972). The induction of temperature-sensitive puffs, on the other hand, has been interpreted to be due to changes of the ATP level or to changes in the oxidized state of a part of the respiratory chain (Leenders et al., 1972b) and to an immigration of certain proteins into the nucleus (Helmsing et al., 1971 ; Helmsing, 1972; Schoon et al., 1973). The possibility that the decrease of the ATP level functions as an inducing signal on the heat-sensitive series of puffs is based on the observation that in experiments with substances that inhibit oxidative phosphorylation, with temperature changes and with anaerobiosis the ATP level must have been lowered and that exogenous ATP inhibits the induction by heat (see Berendes, 1972) and by trinactin (Behnel, unpubl.). The wide occurrence of membrane-bound ATPase, also in the case of the nuclear membrane (Yasuzumi et al., 1966), may indicate a possible role of ATPase in cation and nucleoprotein transport. Since ouabain inhibits the induction of certain puffs by dinactin and puromycin (Schoon et al., 1973), the ATPase involved may be Na÷,K÷-dependent, as seems to be the case in sugar and amino acid transport. The participation of protein carriers, specific for the transported substances, does appear well established (review see Boyer et al., 1972). Energy deprivation of the nuclear membrane might lead to an accumulation of nascent RNA in active chromosome loci as suggested by Yamamoto (1970), Ellgaard et al. (1971); it may also influence the transport of proteins from the cytoplasm into the nucleus as found by Helmsing et al. (1971). The specificity of the puff inductions within the total genetic information and in the course of development is difficult to explain on the basis of our present knowledge. In the experiments reported here, a specific reactivity to different concentrations of the inducing substances is evident and might be interpreted in terms of specific thresholds of different puffs (Lezzi et al., 1972) or by assuming qualitative changes of the effects of the applied substances with changing concentrations. A specific reactivity might be achieved also by mutual inhibition of certain puff series as can be concluded from the low reactivity of heat-sensitive puffs at the time when ecdysone is released (~115 hr) and after incubation with ecdysone. The latter results are in line with the observed regression of other puffs after ecdysone treatment (Ashburner, 1972). Development-specific reactivity in the heat-sensitive group of puffs has been
228
Experimental changes in puffing activity in Drosophila
registered also a f t e r i n c u b a t i o n w i t h v a l i n o m y c i n ( S t e f f e n et al., u n p u b l . ) a n d p u r o m y c i n ( S c h o o n et al., 1 9 7 3 ) in c o n t r a s t to findings o f Ritossa ( 1 9 6 3 , 1 9 6 4 ) a n d o t h e r a u t h o r s w h o r e p o r t e d unspecific reactivity to t e m p e r a t u r e - s h o c k t r e a t m e n t .
ACKNOWLEDGMENT This work was supported by the Deutsche Forschungsgemeinschaft.
REFERENCES Ashburner, M.: Chromosoma (Berl.) 21,398 428 (1967). Ashburner, M.: Chromosoma (Berl.) 3 1 , 3 5 6 - 3 7 6 (1970). Ashburner, M.: In: Developmental Studies on Giant Chromosomes, ed. W. Beerman (Springer Verlag, Berlin, Heidelberg, New York) (1972a). Ashburner, M.: Chromosoma (Berl.) 38,255 281 (1972b). Behnel, H.-J.: unpublished experiments. Berendes, H.D.: In: Developmental Studies on Giant Chromosomes, ed. W. Beerman (Springer Verlag, Berlin, Heidelberg, New York) (1972). Boyer, P.D. and W.L. Klein: In: Membrane Molecular Biology, eds. F. Fox and A. Keith (Sinauer Ass. Inc. Publ., Stanford, Conn.) (1972). Eigen, M. and R. Winkler: In: The Neurosciences, II, ed. F.O. Schmitt (Rockefeller University Press, New York) (1970). EIlgaard, E.G.: Chromosoma (Berl.) 37,417 422 (1972). Ellgaard, E.G. and U. Clever: Chromosoma (Berl.) 36, 6 0 - 7 8 (1971). Graven, S.N., tt.A. Lardy, D. Johnson and A. Rutter: Biochemistry 5, 1729-1734 (1966). Helmsing, P.J. and H.D. Berendes: J. Cell Biol. 5 0 , 8 9 3 - 8 9 6 (1971 ). Helmsing, P.J.: Cell Differentiation 1, 19 24 (1972). Kilbourn, B.T., J.D. Dunitz, L.A.R. Pioda and W. Simon: J. Mol. Biol. 30, 559 563 (1967). Kroeger, H.: Exptl. Cell Res. 4 1 , 6 4 80 (1966). L~uger, P.: Science 178, 2 4 - 3 0 (1972). Leenders, H.J. and P.J.A. Beckers: J. Cell Biol. 55,257-.265 (1972b). Leenders, H.J. and H.D. Berendes: Chromosoma (Berl.) 37,433 444 (1972a). Leenders, tt.J., G.J. Wullems and H.D. Berendes: Exptl. Cell Res. 63, 159-164 (1970). Levitan, H. and J.L. Barker: Science 178, 6 3 - 6 4 (1972). Lezzi, M. and M. Robert: ln: Developmental Studies on Giant Chromosomes, ed. W. Beerman (Springer Verlag, Berlim Heidelberg, New York) (1972). McMurray, W.C.: Arc. Bioch. Biophys. 84,546 (1959). Moore, C. and B.C. Pressman: Biochem. Biophys. Res. Commun. 1 5 , 5 6 2 - 5 6 7 (1964). Nagel, G.: in preparation. Ogata, E. and H. Rasmussen: Biochemistry 5, 57 66 (1966). Rensing, L. and R. Hardeland: Exptl. Cell Res. 73,311 318 (1972). Ritossa, F.: Dros. Inf. Serv. 37,122 (1963). Ritossa, F.: Exptl. Cell Res. 35,601 607 (1964). Robert, M.: Chromosoma (Berl.) 36, 1 - 3 3 (1971). Schoon, H. and L. Rensing: Cell Differentiation, 2, 9 7 - 1 0 6 (1973) Slatcr. E.C.: Proc. Intern. Congr. Biochem. 5th, Moskou (Pergamon Press, New York) (1963). Steffen, K. and L. Rcnsing: unpublished experiments. Yamamoto, H.: Chromosoma (Bcrl.) 32, 171 190 (1970). Yasuzumi, G. and I. Tsubo: E×ptl. Cell Res. 4 3 , 2 8 1 - 2 9 2 (1966).
EFFECTS OF 2,4-DINITROPHENOL
AND DINACTIN ON
HEAT-SENSITIVE AND ECDYSONE-SPECIFIC PUFFS OF
DROSOPHILA
SALIVARY GLAND CHROMOSOMES
IN V I T R O
Ludger RENSING L Zoologisches lnstitut der Universitilt Ggttingen, GOttingen, Western Germany Accepted 10 June 1973
Salivary glands from third instar larvae of Drosophila melanogaster were incubated in vitro with various concentrations of 2,4-dinitrophenol or dinactin and the effects on puff size were measured. At lower concentrations of either substance (10-a M DNP and 10.7 M dinactin) the sizes of the heat-sensitive series of puffs are significantly enlarged. At higher concentrations (10-~ M and 10-4 M) ecdysone-spccific puffs and puffs that appear somewhat later in the course of development are induced whereas the heat-sensitive puffs do not react. A regression of puff size can be observed at higher concentrations in the case of the puffs at 63E and 47BC. The induction of the heat-sensitive series of puffs by dinactin is development-specific: the reactivity of these puffs is maximal at a developmental age of about 117 hr after egg laying (25°C). The normal release of ecdysone during development at about 115 hr as well as the incubation with 1 ~tg/ml or 10/~g/ml ecdysone results in a reduced reactivity to dinactin of puff 87B in particular. A similar effect can be obtained when 10-5 M ouabain is applied.
A certain series of puffs in the salivary glands of Drosophila melanogaster has been shown to be inducible by heat, uncoupling substances (Ritossa, 1963, 1964; Ashburner, 1970; Ellgaard, 1972), and by puromycin and fusidic acid (Schoon et al., 1973). The inductive mechanism may include certain proteins as can be concluded from the latter experiments and the results of Helmsing et al. (1971); it may also include changes in the respiratory metabolism (Leenders et al., 1972a,b). Most substances that uncouple oxidative phosphorylation and induce a specific group of puffs, as for example 2,4-dinitrophenol (DNP), dicumarol, salicylate etc., affect membrane permeabilities for cations, ATPase activity, ATP level and respiration. Which of these effects are of primary importance in the inductive mechanism is far from clear; the same is true for the role of K+-concentrations, cellular and nuclear membrane potentials and their alterations by hormones and cyclic adenosine monophosphate (Kroeger, 1966; Leenders et al., 1970; Lezzi et al., 1972; Rensing et al., 1972). In order to analyse the effects of uncoupling substances in more detail, DNP and an uncoupling antibiotic, dinactin, were tested over a wide range of concentra-
222
Experimental changes in pufft'ng activity in Drosophila
tions. The inductive effect of the antibiotic was registered also under different developmental conditions and under the influence o f ecdysone and ouabain.
MATERIALS AND METHODS Larvae of an inbred stock of D. melanogaster were reared at 25°C under a light-dark cycle of 12:12 hr. At the end of the third instar, the salivary glands were excised, sister glands separated and kept in an artificial medium (modified after Rensing, 1969; Nagel, in preparation). In most cases one of the sister glands was incubated with the substance or antibiotic for 2 hr whereas the other gland served as a control. Squashing of the salivary glands and measurements of the puff size were the same as in experiments described earlier (Rensing et al., 1972): the width of the puff divided by the width of a neighbouring band (puff ratio) of experimental and control glands was taken and the mean and threefold standard error calculated. The following substances were used; 2,4-dinitrophenol (Merck, Darmstadt), dinactin (a gift from Ciba/Geigy, Basel), ouabain (g-strophantin, Merck, Darmstadt), and ecdysone (a gift from Schering, Berlin).
RESULTS Incubation of a sister salivary gland with different concentrations of DNP led to the following results when compared with the other untreated sister gland (fig. 1).
+
15 ~
]25
/~ ,...~, 63Bc
lo-
+ ~i 878
+
"
478C
~
63E
-4 -3 -2 (-1) M Fig. h Effect. of 2,4 dinitrophenol on puff size in salivary gland chromosomes after 2 hr of incubation (third instar larvae, -117 hr). Ordinate: puff ratio of the treated sister gland divided by the puff ratio of the untreated control gland; abscissa: molar concentrations of DNP, (-1) indicates a saturated solution.
L. RENSING
223
j
,
74EF
•
71C
,
1.25 5oCD
1.o
-4
-3
-2
(-I) ~
Fig. 2. Effect of 2,4-dinitrophenol on puff size in salivary gland chromosomes; details as in fig. 1.
Heat-sensitive puffs (Ashburner, 1970), as represented by puffs 63BC and 87B, show maximal increase in puff size at 10-3 M, less increases at 10-4 M, 10-~ M and 10-a M in the case of 63BC and no increase at all at 10-4 M and 10-1 M in the case of 87B. These findings were in line with the earlier observations of Ritossa (1964). A regression of puff size after the incubation period can be noted in 47BC and 63E at concentrations higher than 10-~ M. Development-specific puffs, as represented by puffs that react directly on the release of ecdysone (74EF, 2B, and possibly 50CD) and by puffs activated some hours later in the course of development (71C and 85F), increase their size after incubation with 10-3-10 -1 M DNP significantly (fig. 2). Since the latter effects were observed at very high concentrations and even with a saturated solution of DNP, conditions that bring'about unphysiologic osmotic values, similar experiments with an uncoupling antibiotic, dinactin, were performed. In these experiments the heat-sensitive group of puffs is significantly enlarged at concentrations of 10-9-10-s M dinactin (fig. 3). Maximum size can be observed at 10-7 M in the case of 87B and 03D and at 10-6 M in the case of 63BC. A regression of puffs 47BC and 63E occurs at high concentrations (10 -4 M). The sizes of ecdysone-specific puffs reach a peak at 10-s M (50CD) and 10-4 M (74EF and 2B) whereas later appearing puffs are maximally enlarged at 10-3 M (71C and 85F) as shown in fig. 4. These enlargements must be due to an actual increase in size rather than to an inhibition of the regression process because the control glands do not show any of these development-specific puffs. If these puffs are present at the time when the glands are excised, they are preserved during the incubation in the normal medium. In an attempt to analyse the mechanisms involved in the induced changes of the puffing pattern, it seemed of interest to test the reactivity of the heat-sensitive loci in the course of development. The developmental stage of third instar larvae was
Experimental changes in puffing activity in Drosophila
224
1.75
1.50
1.25
1.o 87B 93D 63E
0:75
-9
-8
-7
-6
-5
-4
-3
M
Fig. 3. Effect of dinactin on p u f f size o f salivary gland c h r o m o s o m e s after 2 hr of incubation; details as in fig. l, abscissa: molar concentrations of dinactin.
74 EF 1.5o
v1? :/.
125
I
2B
71C 85F
5oCD 1.o
-9
-8
-7
-6
-5
-4
-3
M
Fig. 4. Effect o f dinactin on p u f f size in salivary gland c h r o m o s o m e s ; details as in fig. l. abscissa: molar concentrations o f dinactin.
L. RENSING
225
determined following criteria derived from the behaviour of the larvae and the morphology of the salivary glands (Rensing et al., 1972) and also from the puffing pattern (Ashburner, 1967). At the age of 115 hr, characterized by the appearance of ecdysone-specific puffs, the reactivity of heat-sensitive puffs is low when incubated with 10-7 M and 10-6 M dinactin (fig. 5). A maximum reactivity is observed at a time between the release of ecdysone and puparium formation; immediately before puparium formation (120 hr) the reactivity decreases again. If the lower reactivity of the heat-sensitive group of puffs at the developmental age of 115 hr could be related to the release of ecdysone and the induction of ecdysone-specific puffs at that time, addition of ecdysone to the dinactin containing medium should result in a similarly reduced reactivity to the uncoupling substance. This is actually observed in a series of experiments using 1 #g/ml or 10 ~g/ ml ecdysone (table 1), especially significant in the case of puff 87B. The inductive effect of ecdysone, on the other hand, is evident from the significant increase in the size of puff 74EF. No changes at all can be measured in puffs 50CD and 47BC, findings that confirm the validity of the method. Similar results as to the reduced reactivity to dinactin of some puffs are obtained by incubating the salivary glands with the steroid rhamnoside, ouabain (table 1). Ouabain specifically inhibits the activity of the Na ÷, K÷-dependent ATPase. After a
175t
o ~./z°, ~. 1C~o1 °'//'~" ~÷ 125t lo
075
/"
115
87B 63BC 63E 117
'12oh
Fig. 5. Effect of dinactin on puff size in salivary gland chromosomes at different times during development. Ordinate: as in fig. 1, abscissa: developmental age of third instar larvae (Ashburner, 1967). o - - o 10 -7 M, + - - + 10 -6 M dinactin.
226
Experimental changes in puffing activity in Drosophila
Table 1 Effect of the combined incubation (2 hr) with 10-6 M dinactin and 10-5 M ouabain and with 10-7 M dinactin and 1 or 10 ~tg/ml ecdysone as compared to the effect of dinactin alone. Values represent the puff ratios together with the threefold standard error. Puff sites:
63BC
63E
87B
93D
Controls 10-6 M dinactin
1.74_+ 0.13
1.22-+ 0.12
2.02-+ 0.18
2.53_+ 0.36
10-6 M dinactin + 10-5 M ouabain
1.55 _+0.09
1.32 -+ 0.18
1.43 -+ 0.14
2.64 _+0.31
Controls 10-TM dinactin
1.39_+ 0.10
1.18-+ 0.09
1.84_+ 0.18
2.62-+ 0.6
1.30 -+ 0.06
I. 13 -+ 0.06
1.41 -+ 0.16
2.43 _+ 0.5
Puff sites:
47B
50C1)
74EF
2B
Controls 10-6 M dinactin
1.80-+ 0.2
1.69-+ 0.34
1.58-+ 0.24
1.36-+ 0.11
10-6 M dinactin + 10-5 M ouabain
1.70 -+ 0.2
1.70 -+ 0.15
1.64 -+ 0.29
1.40 -+ 0.08
Controls 10-7 M dinactin
1.54 -+ 0.2
1.56 -+ 0.15
1.31 -+ 0.2
1.43 -+ 0.25
10-7 M dinactin + ecdysone (1 or 10 ~g/ml)
1.52-+ 0.13
1.52-+ 0.21
1.73-+ 0.09
1.44 + - 0.18
10 -7 M dinactin
+ ecdysone (1 or 10 ~g/ml)
c o m b i n e d t r e a t m e n t o f t h e glands w i t h b o t h s u b s t a n c e s ( 1 0 -s M o u a b a i n , 10 -6 M d i n a c t i n ) t h e sizes o f puffs 6 3 B C a n d 8 7 B are significantly r e d u c e d as c o m p a r e d w i t h t h o s e glands i n c u b a t e d w i t h d i n a c t i n alone.
DISCUSSION T h e h e a t - s e n s i t i v e series o f puffs ( A s h b u r n e r , 1 9 7 0 ) has b e e n i n d u c e d b y a n u m b e r o f c h e m i c a l u n c o u p l e r s o f o x i d a t i v e p h o s p h o r y l a t i o n , such as 3 × l 0 -3 M s o d i u m azide, 10 -~ M s o d i u m salicylate, 10 -3 M d i c u m a r o l a n d 10 -3 M D N P (Ritossa, 1 9 6 4 ) . T o this list o f s u b s t a n c e s u n c o u p l i n g a n t i b i o t i c s can be a d d e d : 10 - 9 10 -6 M d i n a c t i n , as d e m o n s t r a t e d in this p a p e r , 1 0 - T M m o n a c t i n or t r i n a c t i n (Behnel, u n p u b l . ) a n d 2 × 10 -s M v a l i n o m y c i n ( S t e f f e n et al., u n p u b l . ) w h i c h all i n d u c e t h e same series o f puffs. T h e u n c o u p l i n g m e c h a n i s m a n d o t h e r related effects m a y be similar in t h e case o f u n c o u p l i n g a n t i b i o t i c s a n d c h e m i c a l u n c o u p l e r s : a) a n increase in t h e selective p e r m e a b i l i t y o f m e m b r a n e s for c a t i o n s , especially for K ÷ as it is k n o w n for t h e n o n a c t i n a n t i b i o t i c s ( K i l b o u r n et al., 1 9 6 7 ; Eigen et al., 1970), for v a l i n o m y c i n ( M o o r e et al., 1 9 6 4 ; L~iuger, 1 9 7 2 ) , a n d salicylate ( L e v i t a n et al.,
L. RENSING
227
1972); b) an increase in ATPase activity as demonstrated in the case of DNP and valinomycin (McMurray, 1959), and the nonactin antibiotics (Graven et al., 1966); c) a stimulation of respiration, as for example measured after treatment with DNP (Slater, 1963), nonactin antibiotics (Graven et al., 1966), and valinomycin (Ogata et al., 1966). Some of these effects can also be obtained by temperature changes and reversal of anaerobiosis, conditions that at the same time induce the series of puffs, that respond to the uncoupling substances (reviews, see Berendes, 1972; Ashburner, 1972). Inductions of ecdysone-specific and juvenile hormone-specific puffs have been related to changes of the Na÷/K÷-ratio within the nucleus (Kroeger, 1966; Robert, 1971; Lezzi et al., 1972). The induction of temperature-sensitive puffs, on the other hand, has been interpreted to be due to changes of the ATP level or to changes in the oxidized state of a part of the respiratory chain (Leenders et al., 1972b) and to an immigration of certain proteins into the nucleus (Helmsing et al., 1971 ; Helmsing, 1972; Schoon et al., 1973). The possibility that the decrease of the ATP level functions as an inducing signal on the heat-sensitive series of puffs is based on the observation that in experiments with substances that inhibit oxidative phosphorylation, with temperature changes and with anaerobiosis the ATP level must have been lowered and that exogenous ATP inhibits the induction by heat (see Berendes, 1972) and by trinactin (Behnel, unpubl.). The wide occurrence of membrane-bound ATPase, also in the case of the nuclear membrane (Yasuzumi et al., 1966), may indicate a possible role of ATPase in cation and nucleoprotein transport. Since ouabain inhibits the induction of certain puffs by dinactin and puromycin (Schoon et al., 1973), the ATPase involved may be Na÷,K÷-dependent, as seems to be the case in sugar and amino acid transport. The participation of protein carriers, specific for the transported substances, does appear well established (review see Boyer et al., 1972). Energy deprivation of the nuclear membrane might lead to an accumulation of nascent RNA in active chromosome loci as suggested by Yamamoto (1970), Ellgaard et al. (1971); it may also influence the transport of proteins from the cytoplasm into the nucleus as found by Helmsing et al. (1971). The specificity of the puff inductions within the total genetic information and in the course of development is difficult to explain on the basis of our present knowledge. In the experiments reported here, a specific reactivity to different concentrations of the inducing substances is evident and might be interpreted in terms of specific thresholds of different puffs (Lezzi et al., 1972) or by assuming qualitative changes of the effects of the applied substances with changing concentrations. A specific reactivity might be achieved also by mutual inhibition of certain puff series as can be concluded from the low reactivity of heat-sensitive puffs at the time when ecdysone is released (~115 hr) and after incubation with ecdysone. The latter results are in line with the observed regression of other puffs after ecdysone treatment (Ashburner, 1972). Development-specific reactivity in the heat-sensitive group of puffs has been
228
Experimental changes in puffing activity in Drosophila
registered also a f t e r i n c u b a t i o n w i t h v a l i n o m y c i n ( S t e f f e n et al., u n p u b l . ) a n d p u r o m y c i n ( S c h o o n et al., 1 9 7 3 ) in c o n t r a s t to findings o f Ritossa ( 1 9 6 3 , 1 9 6 4 ) a n d o t h e r a u t h o r s w h o r e p o r t e d unspecific reactivity to t e m p e r a t u r e - s h o c k t r e a t m e n t .
ACKNOWLEDGMENT This work was supported by the Deutsche Forschungsgemeinschaft.
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