Chromosoma (Berl.) 69, 21 -26 (1978)
CHROMOSOMA 9 by Springer-Verlag 1978
Free Genes for rRNAs in the Macronuclear Genome of the Ciliate Stylonychia mytilus Hans Joachim Lipps and Giinther Steinbrfick Institut ffir Biologie III, Abt. Zellbiologie, Universitfit-l-/ibingen, Auf der Morgenstelle 28, D-7400 Ttibingen, Federal Republic of Germany
Abstract. When separated on an agarose gel, macronuclear D N A of the hypotrichous ciliate Stylonychia mytilus gives rise to many well-defined bands ranging in molecular weight from 0.3 • 106 to 14 • 106 dalton. Hybridization of 25 S r R N A , 17 S r R N A or 5 S R N A to such a gel revealed sharp hybridization bands. This suggests that this banding pattern is not an artefact due to nonspecific degradation of macronuclear D N A but that the D N A in the macronucleus of Stylonychia occurs in discrete fragments, each coding for at least one gene. The size of the D N A fragment coding for r R N A was found to be 4.5 • 106 dalton, the fragment coding for 5 S R N A has a molecular weight of 150,000--250,000 dalton.
Introduction
During development of the new macronucleus of hypotrichous ciliates a dramatic qualitative and quantitative change in D N A composition occurs. After mitotic division of the diploid syncaryon formed by the fusion of two haploid gametic micronuclei, a rapid increase in D N A content takes place in the macronuclear anlage, leading to a stage with typical giant chromosomes. These giant chromosomes soon disintegrate and, in Stylonychia mytiIus, more than 95% of the D N A is lost from the nucleus. A second increase in D N A content then leads to the vegetative macronucleus (Ammermann, 1971). While micronuclear D N A and macronuclear anlage D N A contain about 50% repetitive sequences, there are no detectable amounts of these sequences in the vegetative macronucleus (Ammermann et al., 1974). Moreover, the D N A of the micronucleus and the macronuclear anlage always was found to be very large, but it proved difficult to obtain high molecular weight D N A from the macronucleus (Prescott et al., 197l; A m m e r m a n n et al., 1974). While Ammermann et al. (1974) suggested the presence of very active nucleases which digest macronuclear D N A during isolation, Prescott et al. (1973) suggested that these small D N A fragments represent single genes created during development of the new macronucleus.
0009-5915/78/0069/0021/$01.20
22
H.J. Lipps and G. Steinbr/ick
S i n c e t h i s s y s t e m t h e r e f o r e p o s s i b l y p r o v i d e s t h e c h a n c e to a n a l y s e i n d i v i d u a l c o m p l e t e genes we have r e e x a m i n e d this q u e s t i o n with m a c r o n u c l e a r D N A f r o m Stylonychia mytilus. I n this p a p e r w e p r e s e n t e v i d e n c e t h a t at l e a s t r D N A a n d 5 S D N A o c c u r as d e f i n e d D N A f r a g m e n t s .
Material and Methods
Stylonychia mytilus (syngen l) was cultivated in neutral Pringsheim solution and fed faily with the algae Chlorogoniumelongatum (Ammermann et al., 1974). Isolation and purification of macrom~clei and micronuclei was performed as described earlier (Ammermann et al., 1974; Lipps et al., 1974). Macronuclear DNA was isolated using three different procedures: 1) isolated macronuclei were incubated overnight with 400 gg/ml proteinase K (Merck, Darmstadt), 1% SDS, 0.2 M EDTA pH 8 and the DNA was then further purified on a caesium chloride gradient as described by Hennig (1972). 2) Isolated nuclei were lysed with 0.2 M EDTA, 0.15 M NaC1, 1% SDS pH 10, incubated overnight with 400 lag/ml proteinase K and the DNA was further purified by three extractions with phenol-chloroform (1:1 v/v). 3) DNA from whole ceils was prepared by lysing the cells in 0.2 M EDTA, 0.15 M NaC1, 1% SDS pH 10, incubation with 400 gg/ml proteinase K overnight, extraction with phenol-chloroform (1:1) followed by an incubation with 50 !ag/ml RNase A for 4 h, by a proteinase K treatment overnight and by three final extractions with phenolchloroform. Compared to macronuclear DNA, micronuclear DNA represents only about 1% in a preparation from whole cells. All DNA preparations were precipitated with 2 vol. ethanol. Isolation of rRNA followed essentially the technique of Kirby (1965). 25 S and 17 S rRNA from Stylonychia were separated either on a 5-30% linear sucrose gradient in 0.1 M NaC1, 0.02 M sodium acetate, 1 mM EDTA, 0.1% SDS or by electrophoresis on a 2.4% polyacrylamide gel (Loening, 1967) and elution from the gel slices with 0.15 M sodium acetate, i% SDS pH 5. 5 S RNA was purified on a 10% polyacrylamide gel and eluted from the gel slices as described above. Iodination of RNA with 125I (Amersham Buchler) was performed as described by Prensky (1976). Usually a specific activity of about 107 cpm/lag RNA was obtained. DNA was fractionated according to size by electrophoresis on 1.7% or 1% agarose gels in 0.04 M Tris, 0.02 M sodium acetate, 2 mM EDTA pH 7.8 with a constant voltage of 30 V for 16 h. The DNA was stained with ethidium bromide (0.25 gg/ml) and transferred to nitrocellulose filters (Sartorius, 0.151am pore size) according to the technique of Southern (1975) using for the transfer either 10 x SSC for hybridization with rRNA or 20 x SSC for hybridization with 5 S RNA. Hybridization was done in 50% formamide, 2 • SSC at 39~ overnight with an RNA concentration corresponding to about 106 cpm/ml. Filters were then incubated for 1 h with 20 gg/ml RNase A and washed twice with 2 • SSC at 50~ C. As a molecular weight standard 2 DNA digested with restriction endonuclease Eco RI was used. Autoradiography of the filters was done with a Kodak x-omatic film previously sensitized with a short flash (Laskey and Mills, 1975) and with a Kodak x-omatic intensifying screen at - 7 0 ~ C.
Results e l e c t r o p h o r e s e d o n a n a g a r o s e gel m i c r o n u c l e a r D N A o f Stylonychia gives a s h a r p b a n d o n t o p o f t h e gel, d e m o n s t r a t i n g t h a t it is t o o l a r g e t o p e n e t r a t e it (Fig. 1 e). I n c o n t r a s t , m a c r o n u c l e a r D N A r e v e a l s m a n y d e f i n e d b a n d s o n t h e s a m e gel r a n g i n g in m o l e c u l a r w e i g h t f r o m a b o u t 0.3 x 10 6 to 14 • 10 6 d a l t o n w i t h a m a x i m u m a r o u n d 10 6 t o 3 • 106 d a l t o n (Fig. 1 b d). This b a n d i n g p a t t e r n seems to be identical regardless of w h i c h o f the three When
mytilus
methods of DNA
preparations was used.
Free rRNA Genes in the Macronucleus of Stylonychia
23
Fig. la-k. Electrophoretic pattern of Stylonychia DNA separated on a 1.7% agarose gel and hybridization of rRNAs to this gel. a 2 DNA digested with restriction endonuclease Eco RI. The molecular weights of the bands are: 13.7 • 10 6, 4.74 • 10 6, 3.73 • 10 6, 3.48 • 10 6, 3.02 • 10 6 and 2.13 x 10 6 daltons, b Macronuclear DNA isolated according to method 1 (see material and methods). c Macronuclear DNA isolated according to method 2. d Macronuclear DNA isolated from whole celts according to method 3. e Micronuclear DNA of Stylonychia isolated according to method 1. f Hybridisation of Stylonychia 25 S rRNA to macronuclear DNA isolated according to method 1. g Hybridisatiou of 25 S rRNA to macronuclear DNA isolated according to method 2. h Hybridisation of 25 S rRNA to macronuclear DNA isolated according to method 3. i Hybridisation of Stylonychia 25 S + 17 S rRNA to macronuclear DNA isolated according to method 1. k Hybridisatiou of Stylonychia 5 S RNA to macronuclear DNA isolated according to Method 1
I f this b a n d i n g p a t t e r n o f m a c r o n u c l e a r D N A w e r e d u e to n o n s p e c i f i c d e g r a d a t i o n o f D N A d u r i n g i s o l a t i o n , h y b r i d i z a t i o n o f a d e f i n e d R N A species s h o u l d give l a b e l i n g all o v e r the D N A . O n the o t h e r h a n d , if this p a t t e r n is n o t a n a r t e f a c t , h y b r i d i z a t i o n o f s u c h an R N A w o u l d be e x p e c t e d to give a d e f i n e d b a n d . In o r d e r to test these a l t e r n a t i v e s we h y b r i d i z e d ~25I l a b e l e d r R N A a n d 5 S R N A o f Stylonychia to m a c r o n u c l e a r D N A s e p a r a t e d o n a n a g a r o s e gel a n d t h e n t r a n s f e r r e d to n i t r o c e l l u l o s e filters. F i g u r e 1 f i a n d 2 b - c s h o w t h a t r R N A gives a s h a r p b a n d w h e n h y b r i d i z e d to s u c h a filter. T h e p o s i t i o n o f this b a n d was i d e n t i c a l in all D N A p r e p a r a t i o n s used. By c o m p a r i s o n w i t h 2 D N A d i g e s t e d w i t h r e s t r i c t i o n e n d o n u c l e a s e E c o R I the a p p r o x i m a t e m o l e c u l a r w e i g h t o f this b a n d was f o u n d to be 4.5 x 106 d a l t o n . T o see w h e t h e r 25 S a n d 17 S r R N A o c c u r in the s a m e D N A f r a g m e n t , we h y b r i d i z e d s e p a r a t e l y 25 S r R N A (Fig. I f h, Fig. 2 b ) a n d 2 5 S + 1 7 S r R N A
24
H.J. Lipps and G. Steinbrfick
Fig.
2a-c. Hybridisation of r R N A to macronuclear D N A of Stylonychia separated on a 1% agarose gel. a )~ D N A digested with restriction endonuclease Eco RI. b Hybridisation of Stylonychia 25 S r R N A to macronuclear D N A isolated according to method 1 e Hybridisation of Stylonychia 25 S + 17 S r R N A to macronuclear D N A isolated according to method 1
(Figs. l i and 2c) to this D N A . As shown in Figures 1 and 2, both preparations revealed identical bands. As a second R N A probe we used Stylonychia 5 S RNA. Hybridization of this R N A to macronuclear D N A separated on 1.7% agarose gels revealed a very low molecular weight band corresponding to about 200 to 300 base pairs when compared to a limited micrococcal nuclease digest of Stylonychia macronuclei (Lipps and Morris, 1977). Again the position of this band (Fig. 1 k) was identical in different D N A preparations demonstrating that at least r D N A and D N A coding for 5 S R N A occur on discrete D N A fragments in the macronucleus of Stylonychia mytilus.
Discussion
We have shown that macronuclear D N A of Stylonychia mytilus when fractionated on agarose gels gives a typical banding pattern with D N A fragments ranging in molecular weight between 0.3 x 10 6 to 14• 10 6 dalton; a situation very similar to that observed with macronuclear D N A of the related ciliate Oxytricha (Lawn et al., 1978). The banding pattern of Stylonychia macronuclear D N A was identical in all D N A preparations used. Our hybridization experiments using r R N A and 5 S R N A demonstrated that this banding pattern cannot be due to nonspecific degradation of D N A during isolation.
Free rRNA Genes in the Macronucleus of Stylonychia
25
Prescott et al. (1971) have already described the occurrence of very low molecular weight D N A in the macronuclei o f hypotrichous ciliates. They advanced the concept o f "gene-sized D N A pieces" in the macronuclei of these cells (Prescott et al., 1973). However, while Prescott et al. (1971) described the occurrence of only very low molecular weight D N A with an average length of 0.8 lain in the macronuclei of hypotrichous ciliates, A m m e r m a n n et al. (1974) f o u n d that in the macronucleus of Stylonychia mytilus a b o u t half of the D N A molecules are longer than 2.5 ~tm. O u r results show that although large D N A molecules occur in the macronucleus of Stylonychia the average molecular weight ranges f r o m 106 to 3 • 106 dalton. These data are very similar to the data recently published for Oxytricha (Lawn, 1977). Assuming that the r R N A precursor has a molecular weight o f 2 to 2.3 x 106 dalton, the 4.5 x 106 dalton D N A fragment coding for it would be just large e n o u g h to contain one ribosomal gene. Prescott et al. (1973) described a r D N A - c o n t a i n i n g shoulder in a sucrose gradient with an approximate molecular weight o f 4 • 106 dalton in Oxytricha (erroneously called Stylonychia. Lauth et al., 1976). Nonintegrated r D N A was also f o u n d in the macronucleus of the holotrichous ciliate Tetrahymena, but, in contrast to Stylonychia, two ribosomal genes form a palindrome (Karrer and Gall, 1976). The molecular weight of the 5 S D N A fragment with 150,000 to 250,000 dalton would be large enough to code for at least one gene. However, nothing more is k n o w n about this and further w o r k would be required. The observation that micronuclear and macronuclear anlagen D N A is very large suggests that m a c r o n u c l e a r D N A becomes specifically cut during development of the new macronucleus. During breakdown o f the giant c h r o m o s o m e s each band plus its interband becomes enclosed into a vesicle (Kloetzel, 1970) and it seems most likely that during this stage all the D N A not required for vegetative growth becomes degraded, leading to a vegetative macronucleus in which D N A fragments occur, each coding for at least one gene. This system therefore should be most useful for the study of single eucaryotic genes and for possible functional units in the eucaryotic nucleus.
Acknowledgement. This work was supported by the VW-Foundation and the Deutsche Forschungsgemeinschaft.
References Ammermann, D.: Morphology and development of the macronuclei of the ciliates Stylonychia mytilus and Euplotes aediculatus. Chromosoma (Berl.)33, 209-237 (1971) Ammermann, D., Steinbrtick, G., Berger, L. v., Hennig, W. : The development of the macronucleus in the ciliated protozoan Stylonychia mytilus. Chromosoma (Berl.) 45, 401-429 (1974) Hennig, W.: Highly repetitive DNA sequences in the genome of Drosophilahydei. J. molec. Biol. 71, 419-431 (I972) Karrer, K.M., Gall, J.G.: The macronuclear ribosomal DNA of Tetrahymena pyr~fornffs is a palindrome. J. molec. Biol. 104, 421 453 (1976) Kirby, K.S. : Isolation and characterisation of ribosomal ribonucleic acid. Biochem. J. 96, 266 269 (1965) KloetzeI, J.A.: Compartmentalization of the developing macronucleus following conjugation in Stylonychia and Euplotes. J. Cell Biol. 47, 395-407 (1970)
26
H.J. Lipps and G. Steinbr/ick
Laskey, R.A., Mills, A.D.: Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur. J. Biochem. 56, 335-341 (1975) Lauth, M.R., Spear, B.B., Heumann, J., Prescott, D.M. : DNA of ciliated protozoa: DNA sequence diminution during macronuclear development of Oxytricha. Cell 7, 67 74 (1976) Lawn, R.M.: Gene-sized DNA molecules of the Oxytricha macronucleus have the same terminal sequence. Proc. Natl. Acad. Sci. (Wash.) 74, 4325 4328 (1977) Lawn, R.M., Herrick, G., Heumann, J., Prescott, D.M. : Structural organization of the gene-sized pieces of DNA in the ciliate Oxytricha. Cold Spr. Harb. Symp. quant. Biol. 42 (1978) Lipps, H.J., Morris, N.R.: Chromatin structure in the nuclei of the ciliate Stylonychia mytilus. Biochem. biophys. Res. Commun. 74, 230-234 (1977) Lipps, H.J., Sapra, G.R., Ammermann, D.: The histones of the ciliated protozoan Stylonychia mytilus. Chromosoma (Berl.) 45, 273 280 (1974) Loening, U.E. : The determination of the molecular weight of ribonucleic acid by polyacrylamide-gel electrophoresis. Biochem. J. 102, 251 257 (1967) Prensky, W.: The radioiodination of RNA and DNA to high specific activities. In: Methods in cell biology (D.M. Prescott, ed.) 13, 12I 151. New York: Academic Press 1976 Prescott, D.M., Bostock, C.J., Murti, K.G., Lauth, M.R., Gamow, E. : DNA of ciliated protozoa. Chromosoma (Berl.) 34, 355 366 (1971) Prescott, D.M., Murti. K.G., Bostock, C.J.: Genetic apparatus of Stylonychia sp. Nature (Lond.) 242, 597-600 (1973) Southern, E.M. : Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. molec. Biol. 98, 503 517 (1975)
Received April 10 July 8, 1978 / Accepted July 10, 1978 by J.G. Gall Ready for press July 20, 1978
CHROMOSOMA 9 by Springer-Verlag 1978
Free Genes for rRNAs in the Macronuclear Genome of the Ciliate Stylonychia mytilus Hans Joachim Lipps and Giinther Steinbrfick Institut ffir Biologie III, Abt. Zellbiologie, Universitfit-l-/ibingen, Auf der Morgenstelle 28, D-7400 Ttibingen, Federal Republic of Germany
Abstract. When separated on an agarose gel, macronuclear D N A of the hypotrichous ciliate Stylonychia mytilus gives rise to many well-defined bands ranging in molecular weight from 0.3 • 106 to 14 • 106 dalton. Hybridization of 25 S r R N A , 17 S r R N A or 5 S R N A to such a gel revealed sharp hybridization bands. This suggests that this banding pattern is not an artefact due to nonspecific degradation of macronuclear D N A but that the D N A in the macronucleus of Stylonychia occurs in discrete fragments, each coding for at least one gene. The size of the D N A fragment coding for r R N A was found to be 4.5 • 106 dalton, the fragment coding for 5 S R N A has a molecular weight of 150,000--250,000 dalton.
Introduction
During development of the new macronucleus of hypotrichous ciliates a dramatic qualitative and quantitative change in D N A composition occurs. After mitotic division of the diploid syncaryon formed by the fusion of two haploid gametic micronuclei, a rapid increase in D N A content takes place in the macronuclear anlage, leading to a stage with typical giant chromosomes. These giant chromosomes soon disintegrate and, in Stylonychia mytiIus, more than 95% of the D N A is lost from the nucleus. A second increase in D N A content then leads to the vegetative macronucleus (Ammermann, 1971). While micronuclear D N A and macronuclear anlage D N A contain about 50% repetitive sequences, there are no detectable amounts of these sequences in the vegetative macronucleus (Ammermann et al., 1974). Moreover, the D N A of the micronucleus and the macronuclear anlage always was found to be very large, but it proved difficult to obtain high molecular weight D N A from the macronucleus (Prescott et al., 197l; A m m e r m a n n et al., 1974). While Ammermann et al. (1974) suggested the presence of very active nucleases which digest macronuclear D N A during isolation, Prescott et al. (1973) suggested that these small D N A fragments represent single genes created during development of the new macronucleus.
0009-5915/78/0069/0021/$01.20
22
H.J. Lipps and G. Steinbr/ick
S i n c e t h i s s y s t e m t h e r e f o r e p o s s i b l y p r o v i d e s t h e c h a n c e to a n a l y s e i n d i v i d u a l c o m p l e t e genes we have r e e x a m i n e d this q u e s t i o n with m a c r o n u c l e a r D N A f r o m Stylonychia mytilus. I n this p a p e r w e p r e s e n t e v i d e n c e t h a t at l e a s t r D N A a n d 5 S D N A o c c u r as d e f i n e d D N A f r a g m e n t s .
Material and Methods
Stylonychia mytilus (syngen l) was cultivated in neutral Pringsheim solution and fed faily with the algae Chlorogoniumelongatum (Ammermann et al., 1974). Isolation and purification of macrom~clei and micronuclei was performed as described earlier (Ammermann et al., 1974; Lipps et al., 1974). Macronuclear DNA was isolated using three different procedures: 1) isolated macronuclei were incubated overnight with 400 gg/ml proteinase K (Merck, Darmstadt), 1% SDS, 0.2 M EDTA pH 8 and the DNA was then further purified on a caesium chloride gradient as described by Hennig (1972). 2) Isolated nuclei were lysed with 0.2 M EDTA, 0.15 M NaC1, 1% SDS pH 10, incubated overnight with 400 lag/ml proteinase K and the DNA was further purified by three extractions with phenol-chloroform (1:1 v/v). 3) DNA from whole ceils was prepared by lysing the cells in 0.2 M EDTA, 0.15 M NaC1, 1% SDS pH 10, incubation with 400 gg/ml proteinase K overnight, extraction with phenol-chloroform (1:1) followed by an incubation with 50 !ag/ml RNase A for 4 h, by a proteinase K treatment overnight and by three final extractions with phenolchloroform. Compared to macronuclear DNA, micronuclear DNA represents only about 1% in a preparation from whole cells. All DNA preparations were precipitated with 2 vol. ethanol. Isolation of rRNA followed essentially the technique of Kirby (1965). 25 S and 17 S rRNA from Stylonychia were separated either on a 5-30% linear sucrose gradient in 0.1 M NaC1, 0.02 M sodium acetate, 1 mM EDTA, 0.1% SDS or by electrophoresis on a 2.4% polyacrylamide gel (Loening, 1967) and elution from the gel slices with 0.15 M sodium acetate, i% SDS pH 5. 5 S RNA was purified on a 10% polyacrylamide gel and eluted from the gel slices as described above. Iodination of RNA with 125I (Amersham Buchler) was performed as described by Prensky (1976). Usually a specific activity of about 107 cpm/lag RNA was obtained. DNA was fractionated according to size by electrophoresis on 1.7% or 1% agarose gels in 0.04 M Tris, 0.02 M sodium acetate, 2 mM EDTA pH 7.8 with a constant voltage of 30 V for 16 h. The DNA was stained with ethidium bromide (0.25 gg/ml) and transferred to nitrocellulose filters (Sartorius, 0.151am pore size) according to the technique of Southern (1975) using for the transfer either 10 x SSC for hybridization with rRNA or 20 x SSC for hybridization with 5 S RNA. Hybridization was done in 50% formamide, 2 • SSC at 39~ overnight with an RNA concentration corresponding to about 106 cpm/ml. Filters were then incubated for 1 h with 20 gg/ml RNase A and washed twice with 2 • SSC at 50~ C. As a molecular weight standard 2 DNA digested with restriction endonuclease Eco RI was used. Autoradiography of the filters was done with a Kodak x-omatic film previously sensitized with a short flash (Laskey and Mills, 1975) and with a Kodak x-omatic intensifying screen at - 7 0 ~ C.
Results e l e c t r o p h o r e s e d o n a n a g a r o s e gel m i c r o n u c l e a r D N A o f Stylonychia gives a s h a r p b a n d o n t o p o f t h e gel, d e m o n s t r a t i n g t h a t it is t o o l a r g e t o p e n e t r a t e it (Fig. 1 e). I n c o n t r a s t , m a c r o n u c l e a r D N A r e v e a l s m a n y d e f i n e d b a n d s o n t h e s a m e gel r a n g i n g in m o l e c u l a r w e i g h t f r o m a b o u t 0.3 x 10 6 to 14 • 10 6 d a l t o n w i t h a m a x i m u m a r o u n d 10 6 t o 3 • 106 d a l t o n (Fig. 1 b d). This b a n d i n g p a t t e r n seems to be identical regardless of w h i c h o f the three When
mytilus
methods of DNA
preparations was used.
Free rRNA Genes in the Macronucleus of Stylonychia
23
Fig. la-k. Electrophoretic pattern of Stylonychia DNA separated on a 1.7% agarose gel and hybridization of rRNAs to this gel. a 2 DNA digested with restriction endonuclease Eco RI. The molecular weights of the bands are: 13.7 • 10 6, 4.74 • 10 6, 3.73 • 10 6, 3.48 • 10 6, 3.02 • 10 6 and 2.13 x 10 6 daltons, b Macronuclear DNA isolated according to method 1 (see material and methods). c Macronuclear DNA isolated according to method 2. d Macronuclear DNA isolated from whole celts according to method 3. e Micronuclear DNA of Stylonychia isolated according to method 1. f Hybridisation of Stylonychia 25 S rRNA to macronuclear DNA isolated according to method 1. g Hybridisatiou of 25 S rRNA to macronuclear DNA isolated according to method 2. h Hybridisation of 25 S rRNA to macronuclear DNA isolated according to method 3. i Hybridisation of Stylonychia 25 S + 17 S rRNA to macronuclear DNA isolated according to method 1. k Hybridisatiou of Stylonychia 5 S RNA to macronuclear DNA isolated according to Method 1
I f this b a n d i n g p a t t e r n o f m a c r o n u c l e a r D N A w e r e d u e to n o n s p e c i f i c d e g r a d a t i o n o f D N A d u r i n g i s o l a t i o n , h y b r i d i z a t i o n o f a d e f i n e d R N A species s h o u l d give l a b e l i n g all o v e r the D N A . O n the o t h e r h a n d , if this p a t t e r n is n o t a n a r t e f a c t , h y b r i d i z a t i o n o f s u c h an R N A w o u l d be e x p e c t e d to give a d e f i n e d b a n d . In o r d e r to test these a l t e r n a t i v e s we h y b r i d i z e d ~25I l a b e l e d r R N A a n d 5 S R N A o f Stylonychia to m a c r o n u c l e a r D N A s e p a r a t e d o n a n a g a r o s e gel a n d t h e n t r a n s f e r r e d to n i t r o c e l l u l o s e filters. F i g u r e 1 f i a n d 2 b - c s h o w t h a t r R N A gives a s h a r p b a n d w h e n h y b r i d i z e d to s u c h a filter. T h e p o s i t i o n o f this b a n d was i d e n t i c a l in all D N A p r e p a r a t i o n s used. By c o m p a r i s o n w i t h 2 D N A d i g e s t e d w i t h r e s t r i c t i o n e n d o n u c l e a s e E c o R I the a p p r o x i m a t e m o l e c u l a r w e i g h t o f this b a n d was f o u n d to be 4.5 x 106 d a l t o n . T o see w h e t h e r 25 S a n d 17 S r R N A o c c u r in the s a m e D N A f r a g m e n t , we h y b r i d i z e d s e p a r a t e l y 25 S r R N A (Fig. I f h, Fig. 2 b ) a n d 2 5 S + 1 7 S r R N A
24
H.J. Lipps and G. Steinbrfick
Fig.
2a-c. Hybridisation of r R N A to macronuclear D N A of Stylonychia separated on a 1% agarose gel. a )~ D N A digested with restriction endonuclease Eco RI. b Hybridisation of Stylonychia 25 S r R N A to macronuclear D N A isolated according to method 1 e Hybridisation of Stylonychia 25 S + 17 S r R N A to macronuclear D N A isolated according to method 1
(Figs. l i and 2c) to this D N A . As shown in Figures 1 and 2, both preparations revealed identical bands. As a second R N A probe we used Stylonychia 5 S RNA. Hybridization of this R N A to macronuclear D N A separated on 1.7% agarose gels revealed a very low molecular weight band corresponding to about 200 to 300 base pairs when compared to a limited micrococcal nuclease digest of Stylonychia macronuclei (Lipps and Morris, 1977). Again the position of this band (Fig. 1 k) was identical in different D N A preparations demonstrating that at least r D N A and D N A coding for 5 S R N A occur on discrete D N A fragments in the macronucleus of Stylonychia mytilus.
Discussion
We have shown that macronuclear D N A of Stylonychia mytilus when fractionated on agarose gels gives a typical banding pattern with D N A fragments ranging in molecular weight between 0.3 x 10 6 to 14• 10 6 dalton; a situation very similar to that observed with macronuclear D N A of the related ciliate Oxytricha (Lawn et al., 1978). The banding pattern of Stylonychia macronuclear D N A was identical in all D N A preparations used. Our hybridization experiments using r R N A and 5 S R N A demonstrated that this banding pattern cannot be due to nonspecific degradation of D N A during isolation.
Free rRNA Genes in the Macronucleus of Stylonychia
25
Prescott et al. (1971) have already described the occurrence of very low molecular weight D N A in the macronuclei o f hypotrichous ciliates. They advanced the concept o f "gene-sized D N A pieces" in the macronuclei of these cells (Prescott et al., 1973). However, while Prescott et al. (1971) described the occurrence of only very low molecular weight D N A with an average length of 0.8 lain in the macronuclei of hypotrichous ciliates, A m m e r m a n n et al. (1974) f o u n d that in the macronucleus of Stylonychia mytilus a b o u t half of the D N A molecules are longer than 2.5 ~tm. O u r results show that although large D N A molecules occur in the macronucleus of Stylonychia the average molecular weight ranges f r o m 106 to 3 • 106 dalton. These data are very similar to the data recently published for Oxytricha (Lawn, 1977). Assuming that the r R N A precursor has a molecular weight o f 2 to 2.3 x 106 dalton, the 4.5 x 106 dalton D N A fragment coding for it would be just large e n o u g h to contain one ribosomal gene. Prescott et al. (1973) described a r D N A - c o n t a i n i n g shoulder in a sucrose gradient with an approximate molecular weight o f 4 • 106 dalton in Oxytricha (erroneously called Stylonychia. Lauth et al., 1976). Nonintegrated r D N A was also f o u n d in the macronucleus of the holotrichous ciliate Tetrahymena, but, in contrast to Stylonychia, two ribosomal genes form a palindrome (Karrer and Gall, 1976). The molecular weight of the 5 S D N A fragment with 150,000 to 250,000 dalton would be large enough to code for at least one gene. However, nothing more is k n o w n about this and further w o r k would be required. The observation that micronuclear and macronuclear anlagen D N A is very large suggests that m a c r o n u c l e a r D N A becomes specifically cut during development of the new macronucleus. During breakdown o f the giant c h r o m o s o m e s each band plus its interband becomes enclosed into a vesicle (Kloetzel, 1970) and it seems most likely that during this stage all the D N A not required for vegetative growth becomes degraded, leading to a vegetative macronucleus in which D N A fragments occur, each coding for at least one gene. This system therefore should be most useful for the study of single eucaryotic genes and for possible functional units in the eucaryotic nucleus.
Acknowledgement. This work was supported by the VW-Foundation and the Deutsche Forschungsgemeinschaft.
References Ammermann, D.: Morphology and development of the macronuclei of the ciliates Stylonychia mytilus and Euplotes aediculatus. Chromosoma (Berl.)33, 209-237 (1971) Ammermann, D., Steinbrtick, G., Berger, L. v., Hennig, W. : The development of the macronucleus in the ciliated protozoan Stylonychia mytilus. Chromosoma (Berl.) 45, 401-429 (1974) Hennig, W.: Highly repetitive DNA sequences in the genome of Drosophilahydei. J. molec. Biol. 71, 419-431 (I972) Karrer, K.M., Gall, J.G.: The macronuclear ribosomal DNA of Tetrahymena pyr~fornffs is a palindrome. J. molec. Biol. 104, 421 453 (1976) Kirby, K.S. : Isolation and characterisation of ribosomal ribonucleic acid. Biochem. J. 96, 266 269 (1965) KloetzeI, J.A.: Compartmentalization of the developing macronucleus following conjugation in Stylonychia and Euplotes. J. Cell Biol. 47, 395-407 (1970)
26
H.J. Lipps and G. Steinbr/ick
Laskey, R.A., Mills, A.D.: Quantitative film detection of 3H and 14C in polyacrylamide gels by fluorography. Eur. J. Biochem. 56, 335-341 (1975) Lauth, M.R., Spear, B.B., Heumann, J., Prescott, D.M. : DNA of ciliated protozoa: DNA sequence diminution during macronuclear development of Oxytricha. Cell 7, 67 74 (1976) Lawn, R.M.: Gene-sized DNA molecules of the Oxytricha macronucleus have the same terminal sequence. Proc. Natl. Acad. Sci. (Wash.) 74, 4325 4328 (1977) Lawn, R.M., Herrick, G., Heumann, J., Prescott, D.M. : Structural organization of the gene-sized pieces of DNA in the ciliate Oxytricha. Cold Spr. Harb. Symp. quant. Biol. 42 (1978) Lipps, H.J., Morris, N.R.: Chromatin structure in the nuclei of the ciliate Stylonychia mytilus. Biochem. biophys. Res. Commun. 74, 230-234 (1977) Lipps, H.J., Sapra, G.R., Ammermann, D.: The histones of the ciliated protozoan Stylonychia mytilus. Chromosoma (Berl.) 45, 273 280 (1974) Loening, U.E. : The determination of the molecular weight of ribonucleic acid by polyacrylamide-gel electrophoresis. Biochem. J. 102, 251 257 (1967) Prensky, W.: The radioiodination of RNA and DNA to high specific activities. In: Methods in cell biology (D.M. Prescott, ed.) 13, 12I 151. New York: Academic Press 1976 Prescott, D.M., Bostock, C.J., Murti, K.G., Lauth, M.R., Gamow, E. : DNA of ciliated protozoa. Chromosoma (Berl.) 34, 355 366 (1971) Prescott, D.M., Murti. K.G., Bostock, C.J.: Genetic apparatus of Stylonychia sp. Nature (Lond.) 242, 597-600 (1973) Southern, E.M. : Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. molec. Biol. 98, 503 517 (1975)
Received April 10 July 8, 1978 / Accepted July 10, 1978 by J.G. Gall Ready for press July 20, 1978
