J. Mol. Biol. (1977) 112, 353-357
Transcriptional Organization of the Drosophila melanogaster Ribosomal R N A Genes The transcriptional polarity of the 19 S and 26 S ribosomal RNAs of Drosophila melanogaster was investigated using the ultraviolet light mapping technique. The 19 S rRNA was shown to be closer to the promoter than the 26 S rRIqA. In all organisms studied the two larger ribosomal RNA molecules are transcribed as a precursor molecule containing the two rRNAs plus regions of transcribed spacer RNA which vary in size and location from organism to organism. This precursor is then processed by post-transcriptional nucleolytic cleavage into the mature large and small rRNAs (Perry et at., 1970). The transcriptional polarity of the large and small rRNAs on the precursor has been determined for several vertebrate animals (Hackett, 1974; Hackett & Sauerbier, 1975; Reeder et al., 1976; Dawid & Wellauer, 1976), for yeast (van den Bos et al., 1971) and for Escherichia cell (see Pace, 1973, for review). In all cases the smaller rRNA is promoter proximal and the larger rRNA is promoter distal. Drosophila melanogaster rRNA is generated from a precursor molecule having a molecular weight of 2.8 × 106 (Perry et al., 1970) transcribed from transcription units containing one ribosomal precursor cistron per promoter (McKnight & Miller, 1976). However, the ribosomal RNA exhibits several unusual characteristics when compared with the rRNA of other organisms. The mature larger rRNA (26 S) contains a central nick or gap which upon denaturation of the RNA yields two pieces indistinguishable in size from the smaller rRNA (19 S) (Greenberg, 1969; Petri eta/., 1971; Shine & Delgarno, 1973; Jordan, 1975). The 26 S RNA also contains a 2 S RNA hydrogen bonded to it (Jordan et al., 1976). Also Drosophila rRNA has a lower (G ~ C) content than that of other organisms (Hastings & Kirby, 1966). Since Drosophila is evolutionarily quite distant from the other organisms studied and contains rRNA with these unusual characteristics, it was decided to investigate the transcriptional polarity of its ribosomal RNA cistrons. The ultraviolet light mapping technique has proven useful in determining the transcriptional polarity of the rRNA cistrons in E. cell (Haekett & Sauerbier, 1974), mouse L cells (Hackett & Sauerbier, 1975) and HeLa ceils (Hackett, 1974) as well as the order of transcription in bacteriophage (Brautigam & Sauerbier, 1973,1974; Hercules & Sauerbier, 1973,1974) and animal viruses (Ball & White, 1976; Abraham & Banerjee, 1976). We therefore used this technique to determine the polarity of transcription of the 19 S and 26 S rRNAs in D. melanogaster cells. When bacterial and mammalian cells are irradiated with ultraviolet light, transcription terminating lesions are introduced into the DNA. As a result of these lesions, the rate of RNA synthesis in the cells decreases by the function 1 -- e-D/D, where D is the ultraviolet light dose (Sauerbier, 1975,1976). Figure 1 shows that the RNA synthesis in Drosophila cell cultures responds to ultraviolet radiation in the same manner. 353
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Fie. 1. Drosophila melanogazter cells (Schneider's line 2, kindly provided by Alan Blumenthal) were grown in plastic Falcon flasks on Schneider's medium (Pacific Biologicals) supplemented with 15% dialyzed foetal calf serum (Gibco}. The cells were labelled for 20 to 24 h prior to an experiment with 0"2 tzCi [x4C]uridine/ml (35 to 50 mCi/mmol; Schwarz-Mann). The cells at approximately 5 × I0 e to 10V/ml were gently suspended with a rubber policeman and 1.5.ml portions were irradiated with different doses of ultraviolet light with a G.E. G~T4/1 low-pressure mercury lamp at an incident dose of 13 ergs/mm 2 per s 2. The cells were irradiated on a 10-cm diameter watch glass with constant stirring to assure uniform exposure to the ultraviolet light. After irradiation the cells were placed in 15-ml centrifuge tubes and incubated at 25°C in a 25°C shaker bath for 15 rain to allow completion of R N A chains initiated prior to the ultraviolet light treatment. After incubation 50 ~Ci of [3H]uridine/ml (23 Ci/mmol; Schwarz.Mann) was added to each sample and incubation in the 25°C shaker bath was continued for 30 min. The cells were kept in the dark after ultraviolet light irradiation until the end of the labelling period to prevent photoactivated repair of the ultraviolet lesions (Trosko & Wilder, 1973). After labelling, the cells were collected by eentrifugation and resuspended in 1-6 ml of 0-1 M-NaC1, 0.01 M-Tris (pH 7-2), 0-001 ~ - E D T A and lysed at room temperature by the addition of 0-2 ml of 10% sodium dodecyl sulphate, 0-02 ~ - E D T A (pH 7.2). Samples (50-~1) of each lysate were precipitated with 2 ml of 5% triehloroacetic acid, 0.01 M-sodium pyrophosphate, 0.001 M-uridine for 30 min on ice, collected on 25 mm W h a t m a n GDC filters, washed with 10 ml 5% trichloroacetie acid and with 95~o ethanol. The filters were dried and counted with a toluene based P P O / P O P O P scintillation cocktail. The tritium counts in each sample were normalized to the 14C counts in the sample and normalized to the sample receiving no irradiation. The Figure shows individual data points for 4 separate experiments. The curve is the function 1 -- e-D/D, where D = 1 at 11 s. In order to determine the effect of ultraviolet irradiation on the individual rRNA species, Drosophila cells w e r e p r e l a b e l l e d w i t h [14C]uridine, i r r a d i a t e d a n d p u l s e l a b e l l e d w i t h [ S H ] u r i d i n e as d e s c r i b e d in t h e l e g e n d t o F i g u r e 1. T h e R N A w a s i s o l a t e d a n d s u b j e c t e d t o e l e c t r o p h o r e s i s . T h e e l e c t r o p h o r e s i s profiles o f t h e l a b e l l e d R N A a r e s h o w n i n F i g u r e 2 for s e v e r a l d o s e s o f u l t r a v i o l e t i r r a d i a t i o n . T h e z4C.prelabelle d R N A provides both an internal standard for the number of cellular equivalents of RNA a p p l i e d t o e a c h gel a n d m o l e c u l a r w e i g h t m a r k e r s . T h e r a t i o o f t h e t o t a l 14C c o u n t s in
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FIG. 2. Polyaerylamide gel e]ectrophoresis of ultraviolet light irradiated Drosoph//a RNA. Lysates of irradiated cells were prepared as described in the legend to Fig. 1. To purify the RNA, 2.25 g of solid CsC1 was added to each lysate (1.8 ml). This solution was layered onto 1.5 ml of 9.5 molal CsC1 overlayed with mineral oil and centrifuged at 29,000 revs/min for 24 h at 22°C in an SW 50.1 rotor. The pellet and bottom approximately 1 ml of the gradient containing the RNA were pooled, dialysed against 0.01 M-Tris, 0.001 M-EDTA and precipitated with ethanol. The ethanol precipitate was collected by centrifugation, dried under vacuum and dissolved in 0.1 ml of 10% sucrose, 1% sodium dodeeyl sulphate, 0.003 M-EDTA. Electrophoresis of the RNA was performed on either 2-2% polyaerylamide gels or 1"8~o polyaerylamide, 0.5% agarose gels in 0.5-em diam. × 15 cm long glass tubes. The gel buffer was 0.04 MTris.HCl, 0"033 M-sodium acetate, 0.001 ~-EDTA, 0.1% sodium dodecyl sulphate (pH 5.5). Eleetrophoresis was performed at 4 mA/gel for 4 to 6 h. After eleetrophoresis the gel was sliced into 1-mm slices. Two alice samples were dissolved in 0-5 ml of 30% H=O=, 1~o NH4OH and counted in a toluene:triton X100 (2:1), PPO, POPOP scintillation cocktail. The radioactivity profiles shown in the Figure are for 2-2% polyacrylamide gels and have been corrected for channel overlap and background. The non-rRNA background was estimated by the dotted lines shown in the Figure. The ultraviolet light doses are shown in each panel. - - O - - O - - , [14C]Uridine 24 h prelabel; - - 0 - - 0 - - , [3H]uridine 30 min post-ultraviolet light label. t h e 26 S p e a k t o t h e z4C c o u n t s in t h e 19 S p e a k v a r i e d b e t w e e n 1.7 a n d 2-1 for t h e i n d i v i d u a l samples, i n d i c a t i n g t h a t no m o r e t h a n 7 ~ o f t h e d e n a t u r a t i o n l a b i l e 26 S species was b r o k e n d u r i n g t h e i s o l a t i o n p r o c e d u r e in a n y sample. T h e r a d i o a c t i v i t y profiles e x h i b i t t h r e e d i s t i n c t p e a k s s u p e r i m p o s e d on a b r o a d featureless b a c k g r o u n d . T w o o f t h e p e a k s c o m i g r a t e w i t h t h e 19 a n d 26 S 14C-labelled r R N A species. T h e s e were a s s u m e d ~o b e t h e n e w l y s y n t h e s i z e d r R N A molecules. T h e 8H-labeUed 26 S r R N A h a s a n e l e c t r o p h o r e t i c m o b i l i t y s l i g h t l y less t h a n t h a t o f t h e 14C-labelled 26 S
356
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rRNA. This is presumably due to the presence of incompletely processed intermediates to the 26 S rRNA. The third peak has an electrophoretic mobility on polyaerylamide/ agarose gels corresponding to a molecular weight of 2.5 × 106 to 2.8 × 106 which agrees well with the molecular weight assigned to the rRNA precursor. Therefore, this peak was assumed to be the rRNA precursor. In order to quantitate the amount of each ribosomal RNA species the non-ribosomal RNA background was estimated by drawing a smooth curve connecting the regions of the profile on either side of the rRNA peaks. The decrease in synthesis of the individual rRNA species in response to ultraviolet light is shown in Figure 3. Since the synthesis of any given species of RNA should decrease exponentially with dose of ultraviolet light, the individual data points for each species were fitted to straight lines by the method of least-squares. The slopes of the lines were --0.0059, --0.0135 and --0.0137 s-z for the 19 S, 26 S and ribosomal precursor, respectively. The correlation coefficients were --0.78, --0-95 and --0-99, respectively, for the three lines. The absolute value of the slope is directly proportional to the distance of the gene from the promoter. Therefore, we can unambiguously assign the relative order of the rRNA genes on the precursor. The 19 S gene must be promoter proximal since its slope is about 2.3-fold less than that of the 26 S RNA. D. melanogaster is in a different phylum from the other organisms for which the transcriptional polarity of the rI~NA eistrons has been determined. Nevertheless, the small rRNA gene is promoter proximal and the large rRNA gene is promoter distal as in amphibians, mammals, yeast and E. coll. It therefore appears that the transcriptional polarity of the rRNA cistron was established at an early stage in evolution and has been conserved in spite of changes in molecular weight, base composition and nucleolytic processing of the rRNAs.
L E T T E R S TO T H E E D I T O R
357
This research was supported by grants to one of the authors (W. S.) from the National Institutes of Health (5R01-GM-20418) and by grants to Dr David Pettijohn from the U.S. National Science Foundation (GB-43358) and the U.S. Public Health Service (GM-18243). J. C. was supported by a Postdoctoral Fellowship from the National Institutes of Heslt.h (1-F32-HD05098). Department of Biophysics and Genetics University of Colorado Medical Center 4200 E. 9th Avenue Denver, Col. 80262, U.S.A.
JONATHAN CARLSON
GARY OTT WAITER SAUERBIER
Received 26 October 1976, and in revised form 20 J a n u a r y 1977 REFERENCES Abraham, G. & Banerjee, A. K. (1976). Proc. Nat. Aead. Sci., U.S.A. 73, 1504-1508. Ball, L. A. & White, C. N. (1976). Proc. Nat. Acad. Sci., U.S.A. 73, 442-446. Brautigam, A. R. & Sauerbier, W. (1973). J. Virol. 12, 882-886. Brautigam, A. R. & Sauerbier, W. (1974). J. Virol. 13, 1110-1117. Dawid, J. B. & Wellauer, P. K. (1976). Cell, 8, 443-448. Greenberg, J. R. (1969). J. Mol. Biol. 46, 85-98. Hackett, P. B. (1974). Ph.D. Thesis, University of Colorado. Hackett, P. B. & Sauerbicr, W. {1974). Nature (London), 251,639-641. Hackett, P. B. & Sauerbier, W. {1975). J. Mol. Biol. 91, 235-256. Hastings, J. R. B. & Kirby, K. S. (1966). Biochem. J. 1OO, 532. Hercules, K. & Sauerbier, W. (1973). J. Virol. 12, 872-881. Hercules, K. & Sauerbier, W. (1974). J. Virol. 14, 341-348. Jordan, B. R. (1975). J. Mol. Biol. 98, 277-280. Jordan, B. R., Jourdan, R. & Jacq, B. (1976). J. Mol. Biol. 1Ol, 85-105. McKnight, S. L. & Miller, O. J. J r (1976). Cell, 8, 305-319. Pace, N. R. (1973). Bacteriol. Rev. 37, 562-603. Perry, R. P., Cheng, T.-Y., Freed, J. J., Greenberg, J. R., Kelley, D. E. & Tartof, K. D. (1970). Proc. Nat. Acad. Sci., U.S.A. 65, 609-616. Petri, W. H., Fristrom, J. M., Stewart, D. J. & Hanly, E. W. (1971). Mol. Gen. Genet. llO, 245-262. Reeder, R. H., Higashinakagawa, R. & Miller, O. J., J r (1976}. Cell, 8, 449-454. Sauerbier, W. (1975). In Radiation Research, pp. 651-662, Academic Press, New York. Sauerbier, W. (1976). Advan. Radiat. Biol. 6, 49-106. Shine, J. & Delgarno, L. (1973). J. Mol. Biol. 75, 57-72. Trosko, J. E. & Wilder, K. {1973). Genetics, 73, 297-302. van den Bos, R. C., Ketch, J. & Planta, R. J. (1971). Biochim. Biophys. Acta, 232, 494-508.
Transcriptional Organization of the Drosophila melanogaster Ribosomal R N A Genes The transcriptional polarity of the 19 S and 26 S ribosomal RNAs of Drosophila melanogaster was investigated using the ultraviolet light mapping technique. The 19 S rRNA was shown to be closer to the promoter than the 26 S rRIqA. In all organisms studied the two larger ribosomal RNA molecules are transcribed as a precursor molecule containing the two rRNAs plus regions of transcribed spacer RNA which vary in size and location from organism to organism. This precursor is then processed by post-transcriptional nucleolytic cleavage into the mature large and small rRNAs (Perry et at., 1970). The transcriptional polarity of the large and small rRNAs on the precursor has been determined for several vertebrate animals (Hackett, 1974; Hackett & Sauerbier, 1975; Reeder et al., 1976; Dawid & Wellauer, 1976), for yeast (van den Bos et al., 1971) and for Escherichia cell (see Pace, 1973, for review). In all cases the smaller rRNA is promoter proximal and the larger rRNA is promoter distal. Drosophila melanogaster rRNA is generated from a precursor molecule having a molecular weight of 2.8 × 106 (Perry et al., 1970) transcribed from transcription units containing one ribosomal precursor cistron per promoter (McKnight & Miller, 1976). However, the ribosomal RNA exhibits several unusual characteristics when compared with the rRNA of other organisms. The mature larger rRNA (26 S) contains a central nick or gap which upon denaturation of the RNA yields two pieces indistinguishable in size from the smaller rRNA (19 S) (Greenberg, 1969; Petri eta/., 1971; Shine & Delgarno, 1973; Jordan, 1975). The 26 S RNA also contains a 2 S RNA hydrogen bonded to it (Jordan et al., 1976). Also Drosophila rRNA has a lower (G ~ C) content than that of other organisms (Hastings & Kirby, 1966). Since Drosophila is evolutionarily quite distant from the other organisms studied and contains rRNA with these unusual characteristics, it was decided to investigate the transcriptional polarity of its ribosomal RNA cistrons. The ultraviolet light mapping technique has proven useful in determining the transcriptional polarity of the rRNA cistrons in E. cell (Haekett & Sauerbier, 1974), mouse L cells (Hackett & Sauerbier, 1975) and HeLa ceils (Hackett, 1974) as well as the order of transcription in bacteriophage (Brautigam & Sauerbier, 1973,1974; Hercules & Sauerbier, 1973,1974) and animal viruses (Ball & White, 1976; Abraham & Banerjee, 1976). We therefore used this technique to determine the polarity of transcription of the 19 S and 26 S rRNAs in D. melanogaster cells. When bacterial and mammalian cells are irradiated with ultraviolet light, transcription terminating lesions are introduced into the DNA. As a result of these lesions, the rate of RNA synthesis in the cells decreases by the function 1 -- e-D/D, where D is the ultraviolet light dose (Sauerbier, 1975,1976). Figure 1 shows that the RNA synthesis in Drosophila cell cultures responds to ultraviolet radiation in the same manner. 353
354
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Fie. 1. Drosophila melanogazter cells (Schneider's line 2, kindly provided by Alan Blumenthal) were grown in plastic Falcon flasks on Schneider's medium (Pacific Biologicals) supplemented with 15% dialyzed foetal calf serum (Gibco}. The cells were labelled for 20 to 24 h prior to an experiment with 0"2 tzCi [x4C]uridine/ml (35 to 50 mCi/mmol; Schwarz-Mann). The cells at approximately 5 × I0 e to 10V/ml were gently suspended with a rubber policeman and 1.5.ml portions were irradiated with different doses of ultraviolet light with a G.E. G~T4/1 low-pressure mercury lamp at an incident dose of 13 ergs/mm 2 per s 2. The cells were irradiated on a 10-cm diameter watch glass with constant stirring to assure uniform exposure to the ultraviolet light. After irradiation the cells were placed in 15-ml centrifuge tubes and incubated at 25°C in a 25°C shaker bath for 15 rain to allow completion of R N A chains initiated prior to the ultraviolet light treatment. After incubation 50 ~Ci of [3H]uridine/ml (23 Ci/mmol; Schwarz.Mann) was added to each sample and incubation in the 25°C shaker bath was continued for 30 min. The cells were kept in the dark after ultraviolet light irradiation until the end of the labelling period to prevent photoactivated repair of the ultraviolet lesions (Trosko & Wilder, 1973). After labelling, the cells were collected by eentrifugation and resuspended in 1-6 ml of 0-1 M-NaC1, 0.01 M-Tris (pH 7-2), 0-001 ~ - E D T A and lysed at room temperature by the addition of 0-2 ml of 10% sodium dodecyl sulphate, 0-02 ~ - E D T A (pH 7.2). Samples (50-~1) of each lysate were precipitated with 2 ml of 5% triehloroacetic acid, 0.01 M-sodium pyrophosphate, 0.001 M-uridine for 30 min on ice, collected on 25 mm W h a t m a n GDC filters, washed with 10 ml 5% trichloroacetie acid and with 95~o ethanol. The filters were dried and counted with a toluene based P P O / P O P O P scintillation cocktail. The tritium counts in each sample were normalized to the 14C counts in the sample and normalized to the sample receiving no irradiation. The Figure shows individual data points for 4 separate experiments. The curve is the function 1 -- e-D/D, where D = 1 at 11 s. In order to determine the effect of ultraviolet irradiation on the individual rRNA species, Drosophila cells w e r e p r e l a b e l l e d w i t h [14C]uridine, i r r a d i a t e d a n d p u l s e l a b e l l e d w i t h [ S H ] u r i d i n e as d e s c r i b e d in t h e l e g e n d t o F i g u r e 1. T h e R N A w a s i s o l a t e d a n d s u b j e c t e d t o e l e c t r o p h o r e s i s . T h e e l e c t r o p h o r e s i s profiles o f t h e l a b e l l e d R N A a r e s h o w n i n F i g u r e 2 for s e v e r a l d o s e s o f u l t r a v i o l e t i r r a d i a t i o n . T h e z4C.prelabelle d R N A provides both an internal standard for the number of cellular equivalents of RNA a p p l i e d t o e a c h gel a n d m o l e c u l a r w e i g h t m a r k e r s . T h e r a t i o o f t h e t o t a l 14C c o u n t s in
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FIG. 2. Polyaerylamide gel e]ectrophoresis of ultraviolet light irradiated Drosoph//a RNA. Lysates of irradiated cells were prepared as described in the legend to Fig. 1. To purify the RNA, 2.25 g of solid CsC1 was added to each lysate (1.8 ml). This solution was layered onto 1.5 ml of 9.5 molal CsC1 overlayed with mineral oil and centrifuged at 29,000 revs/min for 24 h at 22°C in an SW 50.1 rotor. The pellet and bottom approximately 1 ml of the gradient containing the RNA were pooled, dialysed against 0.01 M-Tris, 0.001 M-EDTA and precipitated with ethanol. The ethanol precipitate was collected by centrifugation, dried under vacuum and dissolved in 0.1 ml of 10% sucrose, 1% sodium dodeeyl sulphate, 0.003 M-EDTA. Electrophoresis of the RNA was performed on either 2-2% polyaerylamide gels or 1"8~o polyaerylamide, 0.5% agarose gels in 0.5-em diam. × 15 cm long glass tubes. The gel buffer was 0.04 MTris.HCl, 0"033 M-sodium acetate, 0.001 ~-EDTA, 0.1% sodium dodecyl sulphate (pH 5.5). Eleetrophoresis was performed at 4 mA/gel for 4 to 6 h. After eleetrophoresis the gel was sliced into 1-mm slices. Two alice samples were dissolved in 0-5 ml of 30% H=O=, 1~o NH4OH and counted in a toluene:triton X100 (2:1), PPO, POPOP scintillation cocktail. The radioactivity profiles shown in the Figure are for 2-2% polyacrylamide gels and have been corrected for channel overlap and background. The non-rRNA background was estimated by the dotted lines shown in the Figure. The ultraviolet light doses are shown in each panel. - - O - - O - - , [14C]Uridine 24 h prelabel; - - 0 - - 0 - - , [3H]uridine 30 min post-ultraviolet light label. t h e 26 S p e a k t o t h e z4C c o u n t s in t h e 19 S p e a k v a r i e d b e t w e e n 1.7 a n d 2-1 for t h e i n d i v i d u a l samples, i n d i c a t i n g t h a t no m o r e t h a n 7 ~ o f t h e d e n a t u r a t i o n l a b i l e 26 S species was b r o k e n d u r i n g t h e i s o l a t i o n p r o c e d u r e in a n y sample. T h e r a d i o a c t i v i t y profiles e x h i b i t t h r e e d i s t i n c t p e a k s s u p e r i m p o s e d on a b r o a d featureless b a c k g r o u n d . T w o o f t h e p e a k s c o m i g r a t e w i t h t h e 19 a n d 26 S 14C-labelled r R N A species. T h e s e were a s s u m e d ~o b e t h e n e w l y s y n t h e s i z e d r R N A molecules. T h e 8H-labeUed 26 S r R N A h a s a n e l e c t r o p h o r e t i c m o b i l i t y s l i g h t l y less t h a n t h a t o f t h e 14C-labelled 26 S
356
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rRNA. This is presumably due to the presence of incompletely processed intermediates to the 26 S rRNA. The third peak has an electrophoretic mobility on polyaerylamide/ agarose gels corresponding to a molecular weight of 2.5 × 106 to 2.8 × 106 which agrees well with the molecular weight assigned to the rRNA precursor. Therefore, this peak was assumed to be the rRNA precursor. In order to quantitate the amount of each ribosomal RNA species the non-ribosomal RNA background was estimated by drawing a smooth curve connecting the regions of the profile on either side of the rRNA peaks. The decrease in synthesis of the individual rRNA species in response to ultraviolet light is shown in Figure 3. Since the synthesis of any given species of RNA should decrease exponentially with dose of ultraviolet light, the individual data points for each species were fitted to straight lines by the method of least-squares. The slopes of the lines were --0.0059, --0.0135 and --0.0137 s-z for the 19 S, 26 S and ribosomal precursor, respectively. The correlation coefficients were --0.78, --0-95 and --0-99, respectively, for the three lines. The absolute value of the slope is directly proportional to the distance of the gene from the promoter. Therefore, we can unambiguously assign the relative order of the rRNA genes on the precursor. The 19 S gene must be promoter proximal since its slope is about 2.3-fold less than that of the 26 S RNA. D. melanogaster is in a different phylum from the other organisms for which the transcriptional polarity of the rI~NA eistrons has been determined. Nevertheless, the small rRNA gene is promoter proximal and the large rRNA gene is promoter distal as in amphibians, mammals, yeast and E. coll. It therefore appears that the transcriptional polarity of the rRNA cistron was established at an early stage in evolution and has been conserved in spite of changes in molecular weight, base composition and nucleolytic processing of the rRNAs.
L E T T E R S TO T H E E D I T O R
357
This research was supported by grants to one of the authors (W. S.) from the National Institutes of Health (5R01-GM-20418) and by grants to Dr David Pettijohn from the U.S. National Science Foundation (GB-43358) and the U.S. Public Health Service (GM-18243). J. C. was supported by a Postdoctoral Fellowship from the National Institutes of Heslt.h (1-F32-HD05098). Department of Biophysics and Genetics University of Colorado Medical Center 4200 E. 9th Avenue Denver, Col. 80262, U.S.A.
JONATHAN CARLSON
GARY OTT WAITER SAUERBIER
Received 26 October 1976, and in revised form 20 J a n u a r y 1977 REFERENCES Abraham, G. & Banerjee, A. K. (1976). Proc. Nat. Aead. Sci., U.S.A. 73, 1504-1508. Ball, L. A. & White, C. N. (1976). Proc. Nat. Acad. Sci., U.S.A. 73, 442-446. Brautigam, A. R. & Sauerbier, W. (1973). J. Virol. 12, 882-886. Brautigam, A. R. & Sauerbier, W. (1974). J. Virol. 13, 1110-1117. Dawid, J. B. & Wellauer, P. K. (1976). Cell, 8, 443-448. Greenberg, J. R. (1969). J. Mol. Biol. 46, 85-98. Hackett, P. B. (1974). Ph.D. Thesis, University of Colorado. Hackett, P. B. & Sauerbicr, W. {1974). Nature (London), 251,639-641. Hackett, P. B. & Sauerbier, W. {1975). J. Mol. Biol. 91, 235-256. Hastings, J. R. B. & Kirby, K. S. (1966). Biochem. J. 1OO, 532. Hercules, K. & Sauerbier, W. (1973). J. Virol. 12, 872-881. Hercules, K. & Sauerbier, W. (1974). J. Virol. 14, 341-348. Jordan, B. R. (1975). J. Mol. Biol. 98, 277-280. Jordan, B. R., Jourdan, R. & Jacq, B. (1976). J. Mol. Biol. 1Ol, 85-105. McKnight, S. L. & Miller, O. J. J r (1976). Cell, 8, 305-319. Pace, N. R. (1973). Bacteriol. Rev. 37, 562-603. Perry, R. P., Cheng, T.-Y., Freed, J. J., Greenberg, J. R., Kelley, D. E. & Tartof, K. D. (1970). Proc. Nat. Acad. Sci., U.S.A. 65, 609-616. Petri, W. H., Fristrom, J. M., Stewart, D. J. & Hanly, E. W. (1971). Mol. Gen. Genet. llO, 245-262. Reeder, R. H., Higashinakagawa, R. & Miller, O. J., J r (1976}. Cell, 8, 449-454. Sauerbier, W. (1975). In Radiation Research, pp. 651-662, Academic Press, New York. Sauerbier, W. (1976). Advan. Radiat. Biol. 6, 49-106. Shine, J. & Delgarno, L. (1973). J. Mol. Biol. 75, 57-72. Trosko, J. E. & Wilder, K. {1973). Genetics, 73, 297-302. van den Bos, R. C., Ketch, J. & Planta, R. J. (1971). Biochim. Biophys. Acta, 232, 494-508.
