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Histone synthesis in Rhynchosciara salivary glands I. Site and timing of synthesis
Cell Differentiation, 4 (1975) 257--263 © North-Holland Publishing Company, Amsterdam -- Printed in The Netherlands
H I S T O N E S Y N T H E S I S IN R H Y N C H O S C I A R A I. S I T E A N D T I M I N G O F S Y N T H E S I S
SALIVARY GLANDS
Manuel TROYANO PUEYO, Maria de FP~TIMA BONALDO and F.J.S. LARA Departamento de Bioqu[mica, Instituto de Qu[mica da Universidade de $5o Paulo, CP 20780 $5o Paulo, Brasil
Accepted 16 July 1975
The site of histone synthesis was studied in polytene cells of the salivary glands of the Rhynchosciara americana (Diptera). It was found that, as is the case in non-polytene
systems, these proteins are synthesized in the cytoplasm in a class of light polysomes which contain 3--4 ribosomes. This class of polyribosomes is most active at about 5 days before pupation when the nuclei are most active in DNA synthesis and the chromosomes of the gland show many open 'DNA puffs'. In R . a m e r i c a n a salivary glands, d u r i n g the last ten days o f the larval life t h e r e is a b u r s t o f D N A synthesis (Lara et al., 1 9 6 7 ; Machado-Santelli et al., 1975) as well as i m p o r t a n t changes in the c h r o m o s o m e m o r p h o l o g y characterized b y t h e a p p e a r a n c e o f t h e so called ' D N A p u f f s ' (Breuer et al., 1955; R u d k i n et al., 1 9 5 7 ) . This burst o f D N A synthesis c o r r e s p o n d s to the initiation o f t h e last r e p l i c a t i o n cycle o f t h e p o l y t e n e c h r o m o s o m e s in the gland, p r i o r to p u p a t i o n . T h e d u r a t i o n o f this cycle is 5--6 days ( C o r d e i r o et al., 1 9 7 3 ; Machado-Santelli et al., 1 9 7 5 ) . T h e unusual length o f the replication phase m a k e s t h e salivary glands o f R . a m e r i c a n a a useful s y s t e m for the s t u d y o f t h e t e m p o r a l relationships b e t w e e n h i s t o n e and D N A synthesis. This p a p e r describes t h e p r e l i m i n a r y e x p e r i m e n t s f o r such s t u d y ; these p r o v i d e i n f o r m a t i o n a b o u t the site o f h i s t o n e synthesis and a b o u t the stage o f larval d e v e l o p m e n t in which the cells o f t h e glands are m o s t active in t h e synthesis o f these proteins. MATERIAL AND METHODS R . a m e r i c a n a larvae were raised u n d e r l a b o r a t o r y c o n d i t i o n s as described b y Lara et al. ( 1 9 6 5 ) . T h e animals used were in the late 4 t h instar which f o r practical p u r p o s e s was divided in several periods m a r k e d b y i m p o r t a n t m o d i f i c a t i o n in larval p h y s i o l o g y ( T e r r a et al., 1 9 7 3 ) . T h e larvae used in t h e e x p e r i m e n t s d e s c r i b e d in this p a p e r were in t h i r d and f i f t h periods o f the 4th
.~bbreviations: PPO, 2,5-diphenyloxazol; POPOP, p-bis[2(5-phenyloxazolylbenzene)|; DTT, dithiothreitol; Tris, tris(hydroxymethyl)aminomethane; TBGLP medium, Terra, Bianchi, Gambarini, Pueyo and Lara's medium.
258
instar, when the DNA puffs are respectively absent or present in the salivary gland chromosomes.
Double-labelling of the salivary glands a) For polyribosomal analysis 100 glands were dissected and incubated for 10 min at 18°C in 750 pl of TBGPL medium (Terra et al., submitted for publication) lacking lysine and tryptophan, but supplemented with 5 tlCi of 4 C-lysine (New England Nuclear, 312 mCi/mMol, 100 pCi/ml) and with 50 pCi of 3 H-tryptophan (New England Nuclear, 5.3 Ci/mMol, 1 mCi/ml). b) For pulse--chase experiments 4 glands from larvae in fifth period were incubated for 15 min at 18°C in 50 pl of the above mentioned medium supplemented with radioactive amino acids as indicated above. At the end of the incubation period, two glands were removed from the medium and fixed in ethanol : chloroform 3 : 1 for 20 min. This procedure avoids solubilization of basic proteins and leaves the glands with a good consistency for microdissection purposes. The other two glands were transferred to fresh TBGPL medium which contained both lysine and t r y p t o p h a n at eight-fold the concentration of radioactive amino acids in the incubation medium, and then chased for 45 min. After chase, the glands were fixed as the other two for subsequent microdissection.
Microdissection of nuclei and cytoplasm of the salivary glands After fixation as indicated above the glands were processed essentially according to Daneholt (1972). The glands were washed 4 times with 70% ethanol containing excess of lysine and t r y p t o p h a n to remove non-specific radioactivity and then maintained for I hr in a solution of ethanol : glycerol I : 1. After this treatment, microdissection was carried out according to Edstr~m (1964) but using a Leitz micromanipulator; only 25 cells from the proximal region of the glands were micro-dissected. The nuclei and the corresponding cytoplasm were carefully collected into separate drops in the oil chamber, under the microscope, and then transferred to scintillation vials containing 0.5 ml of NCS tissue solubilizer (Amersham/Searle Co.). Solubilization was at 50°C for 30 min, and to each vial were added 5 ml of 5 mg PPO; 0.5 mg POPOP per ml of toluene and the radioactivity assayed in a Beckman LS-250 scintillation spectrometer operating with efficiencies of 42% and 94% for 3H and z 4 C respectively. Since these two isotopes were measured simultaneously, care was taken to discount the ~4 C spillover into the 3H window. Usually the spillover of ~4C was 22% in the counting conditions used.
Polyribosomes analysis The detailed process of extraction and sucrose sedimentation of polyri-
259
bosomes from salivary glands is described in the accompanying paper (this Volume, pp. 243--255). The radioactivity assay of the double-labelled nascent peptides in polysomes was made with the caution mentioned in the preceding section. RESULTS
Histones are proteins which characteristically show a high content of basic amino acids such as lysine and arginine b u t lack tryptophan almost completely (Vendrely, 1966; Hnlica, 1967). This fact allowed Robbins et al. (1967) to detect their synthesis in small polyribosomes of HeLa cells by using double-label techniques. In Table I are shown the results of a pulse--chase experiment in which the glands of fifth period larvae were labelled simultaneously with 14 C-lysine and a H-tryptophan. As can be seen, both the cytoplasm and the nuclei of the cells incorporate lysine more efficiently than tryptophan. The amount of lysine that accumulates into the nucleus during the chase period is greater than that of tryptophan, suggesting a preferential transference of basic proteins from cytoplasm to the nucleus during the chase period. In order to determine the class of polysomes which synthesize proteins with high lysine/tryptophan ratio as well as the time at which such syntheses take place, glands of larvae in third and fifth period were labelled simultaneously with ~4 C-lysine and 3 H-tryptophan, their polyribosomes extracted and analyzed in sucrose gradients as indicated in Methods. The results are TABLE I Labelling with a H-tryptophan and 14C-lysine of nuclear and cytoplasmic proteins of salivary gland cells under pulse and chase conditions.
Nucleus Cytoplasm Total cell
3H-tryptophan (CPM)
J 4C.lysine
Pulse(15 min)
Chase(45 min)
Pulse(15 min)
Chase(45 min)
119 1822 1931
152 1671 1823
136 3199 3335
251 3115 3366
Four glands from fifth period larvae were incubated in 50 pl of TBGPL medium lacking lysine and tryptophan but supplemented with 5 ktCi of 14 C-lysine and 50 pCi of 3H-tryptophan. After incorporation for 15 rain at 18°(], two glands were removed and processed for microdissection as described in Methods; the other two glands were transferred to another 50 pl of fresh TBGPL medium containing an excess of cold lysine and tryptophan, and chased for 45 rain at 18°C. After this period the glands were treated as the other two for microdissection. The results were obtained with 25 microdissected cells from the proximal region of the glands. The radioactivity incorporated into nuclei and cytoplasm was assayed as described in Methods and corrected in order to discount the spillover of I 4 C into the 3 H window.
260
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Fig. 1. S i m u l t a n e o u s labelling of n a s c e n t p o l y p e p t i d e s w i t h 3 H - t r y p t o p h a n a n d t 4 C lysine in Rhynchosciara salivary glands f r o m a n i m a l s in t w o d i f f e r e n t d e v e l o p m e n t a l stages. 50 larvae f r o m t h e t h i r d and t h e fifth periods o f t h e 4 t h instar were dissected a n d t h e i r salivary glands s e p a r a t e l y i n c u b a t e d in 750 pl of T B G P L m e d i u m lacking lysine a n d t r y p t o p h a n b u t s u p p l e m e n t e d w i t h 5 pCi of 14C-lysine a n d w i t h 50 /2Ci of 3 H - t r y p t o p h a n , T h e i n c o r p o r a t i o n was allowed to p r o c e e d for 10 m i n at 18°C. A f t e r this period t h e glands were processed in o r d e r to o b t a i n t h e p o s t - m i t o c h o n d r i a l s u p e r n a t a n t s (PMS) 0.3 ml of t h e PMS were layered over 4.5 ml of a linear 1 5 - - 3 0 % sucrose g r a d i e n t s m a d e in 10 mM Tris--HCl; 50 m M KC1; 10 m M Mg(CH3CO2)2 a n d 5 m M D T T at pH 7.4. T h e gradients were s p u n at 6 5 , 0 0 0 g in a SW 5 0 L r o t o r o f a S p i n c o u l t r a c e n t r i f u g e for 2 hr at 4°C. T h e g r a d i e n t s were collected b y p u n c t u r e of t h e b o t t o m of t h e t u b e and t h e o p t i c a l d e n s i t y at 260 n m as well as t h e r a d i o a c t i v i t y assayed as i n d i c a t e d in M e t h o d s . T h e r a d i o a c t i v i t y data were c o r r e c t e d in o r d e r to d i s c o u n t t h e spillover o f 14C into t h e 3 H w i n d o w a n d finally n o r m a l i z e d w i t h respect to t h e m a x i m u m peak in t h e p o l y r i b o s o m e region of e a c h g r a d i e n t to c o m p a r e d i f f e r e n c e s b e t w e e n i n c o r p o r a t i o n of lysine and t r y p t o p h a n along t h e p o l y s o m e profiles f r o m glands of t h i r d (A) a n d fifth (B) p e r i o d larvae. M, p o s i t i o n of t h e m o n o s o m e p e a k ; o - ~, o p t i c a l d e n s i t y at 260 n m ; o--. ---o, normalized counts from 3 H-tryptophan incorporation; nn, n o r m a l i z e d c o u n t s f r o m t 4 C-lysine i n c o r p o r a t i o n ; t h e s h a d e d area i n d i c a t e s t h e region of t h e profile in w h i c h t h e i n c o r p o r a t i o n of lysine is greater t h a n t h a t o f t r y p t o p h a n .
shown in Fig. 1. The radioactivities as ~4 C or 3 H in the polyribosome region of the profile were normalized with respect to the maximum polyribosomal peak in order to detect any divergence of the lysine/tryptophan ratio along the profile. In third period larvae, the curve for lysine falls on that for tryptophan practically along the entire profile. However, in fifth period larvae the lysine curve remains slightly below that for tryptophan except in the region of the
261
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Fig. 2. Effect of h y d r o x y u r e a upon simultaneous 3 H - t r y p t o p h a n and 14C-lysine incorp o r a t i o n into nascent p o l y p e p t i d e s in salivary gland p o l y s o m e s of fifth period larvae. 50 larvae in fifth period were injected with distilled water ( c o n t r o l group) while another 50 larvae of the same stage were injected w i t h 2 pl of a 0.5 M h y d r o x y u r e a solution to inhibit D N A synthesis. The two groups were maintained for 12 hr at 18°C after which their salivary glands were dissected and were incubated separately in 750 ~l o f T B G P L m e d i u m lacking b o t h lysine and t r y p t o p h a n but s u p p l e m e n t e d with 5 pCi of 14 C-lysine and with 50 /2Ci of 3 H - t r y p t o p h a n . A f t e r 10 rain of i n c o r p o r a t i o n at 18°C the glands were processed as described in order to obtain the p o s t - m i t o c h o n d r i a l supernatants. The p o l y r i b o s o m e s of b o t h control (A) and h y d r o x y u r e a treated (B) groups were analyzed in sucrose gradients as in Fig. 1 and the data c o r r e c t e d and normalized in the same manner. See Fig. 1 for e x p l a n a t i o n o f the symbols.
gradient corresponding to 3--4 ribosomes; here the situation is inverted as is indicated by the shaded area in Fig. 1. This divergence suggests that the nascent peptides attached to these polysomes are labelled preferentially with lysine in glands from fifth period larvae. It is important to note that in this stage the DNA synthesis is maximal (Machado-Santelli et al., 1975). Thus, if label with lysine in such light polysomes is equivalent to histone synthesis this would indicate that here we have a correlation between histone and DNA synthesis as has been found for other non-polytene systems (Robbins et al., 1967; Kedes et al., 1969). In order to substantiate this conclusion, we carried out an experiment with fifth period larvae in which DNA synthesis was inhibited with h y d r o x y u r e a (Machado-Santelli et al., 1973). The results obtained are shown in Fig. 2. It can be seen that in hydroxyurea-treated larvae the curves for both lysine and tryptophan run in parallel along the profile and the divergence in the light polyribosome region diminished when
262
compared with that of the control profile obtained from fifth period larvae which were injected with distilled water. This indicates a real coupling between the synthesis of basic proteins in the polyribosomes containing 3--4 ribosomes and DNA synthesis. Moreover, these results strongly suggest that these proteins are histones. DISCUSSION
It is firmly established that in HeLa cells (Robbins et al., 1967) and in early sea urchin blastula (Nemer et al., 1969) the synthesis of histones takes place in the cytoplasm during the replication cycle of the cell. When one takes into consideration the peculiarities of structure and replication of the polytene nuclei as compared to normal diploid nuclei, we could expect differences in the characteristics of histone synthesis in the two systems. In fact incorporation of labelled amino acids by isolated polytene nuclei was detected by several investigators (Sirlin et al., 1962a,b; Ristow et al., 1968; Helmsing, 1970; Cestari et al., 1970). This might indicate that such nuclei are active in protein synthesis which could include histones. The significance of this incorporation remains to be established. Helmsing, however, has inferred from his data that the synthesis of histones in polytene cells from salivary glands of D. hydei larvae should be cytoplasmic (Helmsing, 1971). The results presented in this paper indicate that similarly to what occurs in normal cells, histone synthesis in polytene cells takes place in the cytoplasm. Such synthesis is coupled to DNA synthesis and this was demonstrated both in the case of normal development as well as by inhibition with hydroxyurea which blocks both syntheses. We find that in cells of Rhynchosciara americana salivary glands, histones are synthesized in polysomes containing 3--4 ribosomes (110--130S). Such polysomes are slightly smaller than those reported to be active in histone synthesis in normal diploid cells (Nemer et al., 1969). The experiments presented here do not offer any indication about the nature of this difference. ACKNOWLEDGEMENTS This work was supported by Grants 73/015 and 74/166 from the F u n d a ~ o de Amparo Pesquisa do Estado de S~o Paulo (FAPESP), under the BIOQ-FAPESP Project. One of us (M.T.P.) is a predoctoral fellow from FAPESP.
REFERENCES Breuer, M. and C. Pavan: Chromosoma 7, 3 7 1 - 3 8 6 (1955). Cestari, A.N. and C. Pavan: J. Cell Biol. 47, 32-A (1970). Cordeiro, M. and R. Meneghini: J. Mol. Biol. 7 8 , 2 6 1 - - 2 7 4 (1973). Daneholt, B.: Nature New Biol. 240, 229--232 (1972).
263 Edstr6m, J.E.: In: Methods in Cell Biology, VI, ed. D. Prescott (Academic Press, New York) pp. 417 477 (1964) Helmsing, P.J.: Biochem. Biophys. Acta 224, 587 (1970). Helmsing, P.J.: Biochem. Biophys. Acta 2 3 2 , 7 3 3 - - 7 5 5 (1971). Hnlica, L.: Prog. Nucleic Acid Res. Mol. Biol. 7, 25--106 (1967). Kedes, H., P.R. Gross, G. Cognetti and A.L. Hunter: J. Mol. Biol. 45, 337---351 (1969). Lara, F.J.S., H. Tamaki and C. Pavan: Am. Nat. 9 9 , 1 8 9 - - 1 9 1 (1965). Lara, F.J.S. and F.M. Hollander: Nat. Cancer Inst. Monogr. 27,235--242 (1967). Machado-Santelli, G.M. and R. Basile: Genetics 74, 168 (1973). Machado-Santelli, G.M. and R. Basile: Cienc. Cult. Silo Paulo 27,167 174 (1975). Nemer, M. and D.T. Lindsay: Biochem. Biophys. Res. Commun. 3 5 , 1 5 6 - 160 (1969). Ristow, H. and S. Arendes: Biochem. Biophys. Acta 1 5 7 , 1 7 8 - 186 (1968). Robbins, E. and T.W. Borun: Proc. Nat. Acad. Sci. U.S.A. 5 7 , 4 0 9 - 4 1 6 (1967). Rudkin, G.T. and S.L. Corlette:Proc. Nat. Acad. Sci. U.S.A. 4 3 , 9 6 4 968 (1957). Sirlin, J.L. and N.A. Schor: Exp. Cell Res. 27,165--167 (1962a). Sirlin, J.L. and N.A. Schor: Exp. Cell Res. 2 7 , 3 6 3 - 3 6 6 ('1962b). Terra, W.R., A.G. de Bianchi, A.G. Gambarini and F.J.S. Lara: J. Insect Physiol. 19, 2 0 9 7 - 2 1 0 6 (1973). Vendrely, R. and C. Vendrely: Protoplasmatologia 5, 1 - 8 8 (1966).
H I S T O N E S Y N T H E S I S IN R H Y N C H O S C I A R A I. S I T E A N D T I M I N G O F S Y N T H E S I S
SALIVARY GLANDS
Manuel TROYANO PUEYO, Maria de FP~TIMA BONALDO and F.J.S. LARA Departamento de Bioqu[mica, Instituto de Qu[mica da Universidade de $5o Paulo, CP 20780 $5o Paulo, Brasil
Accepted 16 July 1975
The site of histone synthesis was studied in polytene cells of the salivary glands of the Rhynchosciara americana (Diptera). It was found that, as is the case in non-polytene
systems, these proteins are synthesized in the cytoplasm in a class of light polysomes which contain 3--4 ribosomes. This class of polyribosomes is most active at about 5 days before pupation when the nuclei are most active in DNA synthesis and the chromosomes of the gland show many open 'DNA puffs'. In R . a m e r i c a n a salivary glands, d u r i n g the last ten days o f the larval life t h e r e is a b u r s t o f D N A synthesis (Lara et al., 1 9 6 7 ; Machado-Santelli et al., 1975) as well as i m p o r t a n t changes in the c h r o m o s o m e m o r p h o l o g y characterized b y t h e a p p e a r a n c e o f t h e so called ' D N A p u f f s ' (Breuer et al., 1955; R u d k i n et al., 1 9 5 7 ) . This burst o f D N A synthesis c o r r e s p o n d s to the initiation o f t h e last r e p l i c a t i o n cycle o f t h e p o l y t e n e c h r o m o s o m e s in the gland, p r i o r to p u p a t i o n . T h e d u r a t i o n o f this cycle is 5--6 days ( C o r d e i r o et al., 1 9 7 3 ; Machado-Santelli et al., 1 9 7 5 ) . T h e unusual length o f the replication phase m a k e s t h e salivary glands o f R . a m e r i c a n a a useful s y s t e m for the s t u d y o f t h e t e m p o r a l relationships b e t w e e n h i s t o n e and D N A synthesis. This p a p e r describes t h e p r e l i m i n a r y e x p e r i m e n t s f o r such s t u d y ; these p r o v i d e i n f o r m a t i o n a b o u t the site o f h i s t o n e synthesis and a b o u t the stage o f larval d e v e l o p m e n t in which the cells o f t h e glands are m o s t active in t h e synthesis o f these proteins. MATERIAL AND METHODS R . a m e r i c a n a larvae were raised u n d e r l a b o r a t o r y c o n d i t i o n s as described b y Lara et al. ( 1 9 6 5 ) . T h e animals used were in the late 4 t h instar which f o r practical p u r p o s e s was divided in several periods m a r k e d b y i m p o r t a n t m o d i f i c a t i o n in larval p h y s i o l o g y ( T e r r a et al., 1 9 7 3 ) . T h e larvae used in t h e e x p e r i m e n t s d e s c r i b e d in this p a p e r were in t h i r d and f i f t h periods o f the 4th
.~bbreviations: PPO, 2,5-diphenyloxazol; POPOP, p-bis[2(5-phenyloxazolylbenzene)|; DTT, dithiothreitol; Tris, tris(hydroxymethyl)aminomethane; TBGLP medium, Terra, Bianchi, Gambarini, Pueyo and Lara's medium.
258
instar, when the DNA puffs are respectively absent or present in the salivary gland chromosomes.
Double-labelling of the salivary glands a) For polyribosomal analysis 100 glands were dissected and incubated for 10 min at 18°C in 750 pl of TBGPL medium (Terra et al., submitted for publication) lacking lysine and tryptophan, but supplemented with 5 tlCi of 4 C-lysine (New England Nuclear, 312 mCi/mMol, 100 pCi/ml) and with 50 pCi of 3 H-tryptophan (New England Nuclear, 5.3 Ci/mMol, 1 mCi/ml). b) For pulse--chase experiments 4 glands from larvae in fifth period were incubated for 15 min at 18°C in 50 pl of the above mentioned medium supplemented with radioactive amino acids as indicated above. At the end of the incubation period, two glands were removed from the medium and fixed in ethanol : chloroform 3 : 1 for 20 min. This procedure avoids solubilization of basic proteins and leaves the glands with a good consistency for microdissection purposes. The other two glands were transferred to fresh TBGPL medium which contained both lysine and t r y p t o p h a n at eight-fold the concentration of radioactive amino acids in the incubation medium, and then chased for 45 min. After chase, the glands were fixed as the other two for subsequent microdissection.
Microdissection of nuclei and cytoplasm of the salivary glands After fixation as indicated above the glands were processed essentially according to Daneholt (1972). The glands were washed 4 times with 70% ethanol containing excess of lysine and t r y p t o p h a n to remove non-specific radioactivity and then maintained for I hr in a solution of ethanol : glycerol I : 1. After this treatment, microdissection was carried out according to Edstr~m (1964) but using a Leitz micromanipulator; only 25 cells from the proximal region of the glands were micro-dissected. The nuclei and the corresponding cytoplasm were carefully collected into separate drops in the oil chamber, under the microscope, and then transferred to scintillation vials containing 0.5 ml of NCS tissue solubilizer (Amersham/Searle Co.). Solubilization was at 50°C for 30 min, and to each vial were added 5 ml of 5 mg PPO; 0.5 mg POPOP per ml of toluene and the radioactivity assayed in a Beckman LS-250 scintillation spectrometer operating with efficiencies of 42% and 94% for 3H and z 4 C respectively. Since these two isotopes were measured simultaneously, care was taken to discount the ~4 C spillover into the 3H window. Usually the spillover of ~4C was 22% in the counting conditions used.
Polyribosomes analysis The detailed process of extraction and sucrose sedimentation of polyri-
259
bosomes from salivary glands is described in the accompanying paper (this Volume, pp. 243--255). The radioactivity assay of the double-labelled nascent peptides in polysomes was made with the caution mentioned in the preceding section. RESULTS
Histones are proteins which characteristically show a high content of basic amino acids such as lysine and arginine b u t lack tryptophan almost completely (Vendrely, 1966; Hnlica, 1967). This fact allowed Robbins et al. (1967) to detect their synthesis in small polyribosomes of HeLa cells by using double-label techniques. In Table I are shown the results of a pulse--chase experiment in which the glands of fifth period larvae were labelled simultaneously with 14 C-lysine and a H-tryptophan. As can be seen, both the cytoplasm and the nuclei of the cells incorporate lysine more efficiently than tryptophan. The amount of lysine that accumulates into the nucleus during the chase period is greater than that of tryptophan, suggesting a preferential transference of basic proteins from cytoplasm to the nucleus during the chase period. In order to determine the class of polysomes which synthesize proteins with high lysine/tryptophan ratio as well as the time at which such syntheses take place, glands of larvae in third and fifth period were labelled simultaneously with ~4 C-lysine and 3 H-tryptophan, their polyribosomes extracted and analyzed in sucrose gradients as indicated in Methods. The results are TABLE I Labelling with a H-tryptophan and 14C-lysine of nuclear and cytoplasmic proteins of salivary gland cells under pulse and chase conditions.
Nucleus Cytoplasm Total cell
3H-tryptophan (CPM)
J 4C.lysine
Pulse(15 min)
Chase(45 min)
Pulse(15 min)
Chase(45 min)
119 1822 1931
152 1671 1823
136 3199 3335
251 3115 3366
Four glands from fifth period larvae were incubated in 50 pl of TBGPL medium lacking lysine and tryptophan but supplemented with 5 ktCi of 14 C-lysine and 50 pCi of 3H-tryptophan. After incorporation for 15 rain at 18°(], two glands were removed and processed for microdissection as described in Methods; the other two glands were transferred to another 50 pl of fresh TBGPL medium containing an excess of cold lysine and tryptophan, and chased for 45 rain at 18°C. After this period the glands were treated as the other two for microdissection. The results were obtained with 25 microdissected cells from the proximal region of the glands. The radioactivity incorporated into nuclei and cytoplasm was assayed as described in Methods and corrected in order to discount the spillover of I 4 C into the 3 H window.
260
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Fig. 1. S i m u l t a n e o u s labelling of n a s c e n t p o l y p e p t i d e s w i t h 3 H - t r y p t o p h a n a n d t 4 C lysine in Rhynchosciara salivary glands f r o m a n i m a l s in t w o d i f f e r e n t d e v e l o p m e n t a l stages. 50 larvae f r o m t h e t h i r d and t h e fifth periods o f t h e 4 t h instar were dissected a n d t h e i r salivary glands s e p a r a t e l y i n c u b a t e d in 750 pl of T B G P L m e d i u m lacking lysine a n d t r y p t o p h a n b u t s u p p l e m e n t e d w i t h 5 pCi of 14C-lysine a n d w i t h 50 /2Ci of 3 H - t r y p t o p h a n , T h e i n c o r p o r a t i o n was allowed to p r o c e e d for 10 m i n at 18°C. A f t e r this period t h e glands were processed in o r d e r to o b t a i n t h e p o s t - m i t o c h o n d r i a l s u p e r n a t a n t s (PMS) 0.3 ml of t h e PMS were layered over 4.5 ml of a linear 1 5 - - 3 0 % sucrose g r a d i e n t s m a d e in 10 mM Tris--HCl; 50 m M KC1; 10 m M Mg(CH3CO2)2 a n d 5 m M D T T at pH 7.4. T h e gradients were s p u n at 6 5 , 0 0 0 g in a SW 5 0 L r o t o r o f a S p i n c o u l t r a c e n t r i f u g e for 2 hr at 4°C. T h e g r a d i e n t s were collected b y p u n c t u r e of t h e b o t t o m of t h e t u b e and t h e o p t i c a l d e n s i t y at 260 n m as well as t h e r a d i o a c t i v i t y assayed as i n d i c a t e d in M e t h o d s . T h e r a d i o a c t i v i t y data were c o r r e c t e d in o r d e r to d i s c o u n t t h e spillover o f 14C into t h e 3 H w i n d o w a n d finally n o r m a l i z e d w i t h respect to t h e m a x i m u m peak in t h e p o l y r i b o s o m e region of e a c h g r a d i e n t to c o m p a r e d i f f e r e n c e s b e t w e e n i n c o r p o r a t i o n of lysine and t r y p t o p h a n along t h e p o l y s o m e profiles f r o m glands of t h i r d (A) a n d fifth (B) p e r i o d larvae. M, p o s i t i o n of t h e m o n o s o m e p e a k ; o - ~, o p t i c a l d e n s i t y at 260 n m ; o--. ---o, normalized counts from 3 H-tryptophan incorporation; nn, n o r m a l i z e d c o u n t s f r o m t 4 C-lysine i n c o r p o r a t i o n ; t h e s h a d e d area i n d i c a t e s t h e region of t h e profile in w h i c h t h e i n c o r p o r a t i o n of lysine is greater t h a n t h a t o f t r y p t o p h a n .
shown in Fig. 1. The radioactivities as ~4 C or 3 H in the polyribosome region of the profile were normalized with respect to the maximum polyribosomal peak in order to detect any divergence of the lysine/tryptophan ratio along the profile. In third period larvae, the curve for lysine falls on that for tryptophan practically along the entire profile. However, in fifth period larvae the lysine curve remains slightly below that for tryptophan except in the region of the
261
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Fig. 2. Effect of h y d r o x y u r e a upon simultaneous 3 H - t r y p t o p h a n and 14C-lysine incorp o r a t i o n into nascent p o l y p e p t i d e s in salivary gland p o l y s o m e s of fifth period larvae. 50 larvae in fifth period were injected with distilled water ( c o n t r o l group) while another 50 larvae of the same stage were injected w i t h 2 pl of a 0.5 M h y d r o x y u r e a solution to inhibit D N A synthesis. The two groups were maintained for 12 hr at 18°C after which their salivary glands were dissected and were incubated separately in 750 ~l o f T B G P L m e d i u m lacking b o t h lysine and t r y p t o p h a n but s u p p l e m e n t e d with 5 pCi of 14 C-lysine and with 50 /2Ci of 3 H - t r y p t o p h a n . A f t e r 10 rain of i n c o r p o r a t i o n at 18°C the glands were processed as described in order to obtain the p o s t - m i t o c h o n d r i a l supernatants. The p o l y r i b o s o m e s of b o t h control (A) and h y d r o x y u r e a treated (B) groups were analyzed in sucrose gradients as in Fig. 1 and the data c o r r e c t e d and normalized in the same manner. See Fig. 1 for e x p l a n a t i o n o f the symbols.
gradient corresponding to 3--4 ribosomes; here the situation is inverted as is indicated by the shaded area in Fig. 1. This divergence suggests that the nascent peptides attached to these polysomes are labelled preferentially with lysine in glands from fifth period larvae. It is important to note that in this stage the DNA synthesis is maximal (Machado-Santelli et al., 1975). Thus, if label with lysine in such light polysomes is equivalent to histone synthesis this would indicate that here we have a correlation between histone and DNA synthesis as has been found for other non-polytene systems (Robbins et al., 1967; Kedes et al., 1969). In order to substantiate this conclusion, we carried out an experiment with fifth period larvae in which DNA synthesis was inhibited with h y d r o x y u r e a (Machado-Santelli et al., 1973). The results obtained are shown in Fig. 2. It can be seen that in hydroxyurea-treated larvae the curves for both lysine and tryptophan run in parallel along the profile and the divergence in the light polyribosome region diminished when
262
compared with that of the control profile obtained from fifth period larvae which were injected with distilled water. This indicates a real coupling between the synthesis of basic proteins in the polyribosomes containing 3--4 ribosomes and DNA synthesis. Moreover, these results strongly suggest that these proteins are histones. DISCUSSION
It is firmly established that in HeLa cells (Robbins et al., 1967) and in early sea urchin blastula (Nemer et al., 1969) the synthesis of histones takes place in the cytoplasm during the replication cycle of the cell. When one takes into consideration the peculiarities of structure and replication of the polytene nuclei as compared to normal diploid nuclei, we could expect differences in the characteristics of histone synthesis in the two systems. In fact incorporation of labelled amino acids by isolated polytene nuclei was detected by several investigators (Sirlin et al., 1962a,b; Ristow et al., 1968; Helmsing, 1970; Cestari et al., 1970). This might indicate that such nuclei are active in protein synthesis which could include histones. The significance of this incorporation remains to be established. Helmsing, however, has inferred from his data that the synthesis of histones in polytene cells from salivary glands of D. hydei larvae should be cytoplasmic (Helmsing, 1971). The results presented in this paper indicate that similarly to what occurs in normal cells, histone synthesis in polytene cells takes place in the cytoplasm. Such synthesis is coupled to DNA synthesis and this was demonstrated both in the case of normal development as well as by inhibition with hydroxyurea which blocks both syntheses. We find that in cells of Rhynchosciara americana salivary glands, histones are synthesized in polysomes containing 3--4 ribosomes (110--130S). Such polysomes are slightly smaller than those reported to be active in histone synthesis in normal diploid cells (Nemer et al., 1969). The experiments presented here do not offer any indication about the nature of this difference. ACKNOWLEDGEMENTS This work was supported by Grants 73/015 and 74/166 from the F u n d a ~ o de Amparo Pesquisa do Estado de S~o Paulo (FAPESP), under the BIOQ-FAPESP Project. One of us (M.T.P.) is a predoctoral fellow from FAPESP.
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263 Edstr6m, J.E.: In: Methods in Cell Biology, VI, ed. D. Prescott (Academic Press, New York) pp. 417 477 (1964) Helmsing, P.J.: Biochem. Biophys. Acta 224, 587 (1970). Helmsing, P.J.: Biochem. Biophys. Acta 2 3 2 , 7 3 3 - - 7 5 5 (1971). Hnlica, L.: Prog. Nucleic Acid Res. Mol. Biol. 7, 25--106 (1967). Kedes, H., P.R. Gross, G. Cognetti and A.L. Hunter: J. Mol. Biol. 45, 337---351 (1969). Lara, F.J.S., H. Tamaki and C. Pavan: Am. Nat. 9 9 , 1 8 9 - - 1 9 1 (1965). Lara, F.J.S. and F.M. Hollander: Nat. Cancer Inst. Monogr. 27,235--242 (1967). Machado-Santelli, G.M. and R. Basile: Genetics 74, 168 (1973). Machado-Santelli, G.M. and R. Basile: Cienc. Cult. Silo Paulo 27,167 174 (1975). Nemer, M. and D.T. Lindsay: Biochem. Biophys. Res. Commun. 3 5 , 1 5 6 - 160 (1969). Ristow, H. and S. Arendes: Biochem. Biophys. Acta 1 5 7 , 1 7 8 - 186 (1968). Robbins, E. and T.W. Borun: Proc. Nat. Acad. Sci. U.S.A. 5 7 , 4 0 9 - 4 1 6 (1967). Rudkin, G.T. and S.L. Corlette:Proc. Nat. Acad. Sci. U.S.A. 4 3 , 9 6 4 968 (1957). Sirlin, J.L. and N.A. Schor: Exp. Cell Res. 27,165--167 (1962a). Sirlin, J.L. and N.A. Schor: Exp. Cell Res. 2 7 , 3 6 3 - 3 6 6 ('1962b). Terra, W.R., A.G. de Bianchi, A.G. Gambarini and F.J.S. Lara: J. Insect Physiol. 19, 2 0 9 7 - 2 1 0 6 (1973). Vendrely, R. and C. Vendrely: Protoplasmatologia 5, 1 - 8 8 (1966).