THE SYNTHESIS OF 5s RNA AND ITS RELATIONSHIP TO 18s AND 28s RIBOSOMAL RNA IN THE BOBBED MUTANTS O F DROSOPHILA MELANOGASTER JAG MOHAN
Department of Genetics, Haryana Agricultural University, Hissar-125004,
India
Manuscript received February 3, 1975 Revised copy received June 15, 1975 ABSTRACT
Ribosomes contain one molecule each of 5S, 18s and 28s RNA. In Drosophila melanogaster although the genes for 18s 28s are physically separated from the 5s RNA genes, the multiplicity of various ribosomal RNA genes is roughly the same. Thus a coordinate synthesis of these three molecules might seem feasible. This problem has been approached by determining the molar ratios of various R N A s i n ovaries and in adult flies. In ovaries there is a slight excess of 5s RNA molecules over other rRNA's, but in adult flies no such differences exist. Bobbed mutants also have the same molar ratios as wild-type flies. Results on 5s RNA synthesis in both in vitro and in vivo studies show that it is reduced in coordination with 18s 28s rRNA in the bobbed mutants of Drosophila melanogaster. Various possibilities are discussed in considering the implications of these results.
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RIBOSOMES contain one molecule each of three distinct types of ribonucleic acid (rRNA) conventionally known by their sedimentation values as 28S, 18s and 5s in higher organisms. Genes for 28s and 18s rRNA are localized a t CHIPCHASE and SPIERS1971). the sites of the nucleolar organizers (BIRNSTIEL, In Drosophila melanogaster 5 s RNA genes are clustered away from nucleolar and STEFFENSEN organizer at the 56F locus of the second chromosome (WIMBER STEFFENSEN and HUGHES 1973) and their number is in near 1970; PRENSKY, parity to 18s and 28s rRNA genes (TARTOF and PERRY 1970; QUINCEY1971). This differs sharply from the situation observed in other eukaryotes where genes and ATTARDI for 5s RNA are spread over several chromosomes (ALONI,HALTEN 1971; PARDUE, BROWNand BIRNSTIEL1973; STEFFENSEN and DUFFEY1974) and there is an excess of these genes over 18s and 28s rRNA genes (BROWN and WEBER 1968; QUINCEYand WILSON 1969; AMALDIand BUONGIORNO-NARDELLI 1971; BIRNSTIEL,SELLSand PURDOM 1972; BROWNand SUGIMOTO 1973). In most cells three types of rRNA's are present in equimolar amounts (DENIS, WEGNEZ and MAIRY1973). However, 5s RNA synthesis is not always coordinated (ABE and YAMANA 1971). During early oogenesis in Xenopus, there is 1971) . I n mamnon-coordinated synthesis and accumulation of 5s RNA (FORD malian cells, 5s RNA synthesis continues in the absence of 18s and 28s synthesis Genetics 81: 723-738 Decembei, 1975
724
J. MOHAN
(PERRY and KELLY1968) and there is an excess of 5s RNA synthesis (LIEBOWEINBERG and PENMAN 1973). In the present study 5s RNA synthesis and its rela tionship to 18s -I-28s rRNA in Drosophila melanogaster has been studied. It is possible by genetic manipulation to alter the number of 18s 28s rRNA genes in this organism (RITOSSA and SPIEGELMAN 1965; RITOSSA,ATWOOD and SPIEGELMAN 1966). Drosophila geno-
WITZ.
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types containing less than 130 such genes manifest a bobbed phenotype characterized by slow development, smaIl bristles and etching of the abdominal tergites. We have shown (MOHAN and RITOSSA1970) that the rate of 18s -I-28s rRNA synthesis is reduced in bobbed flies, but they have the same RNA/DNA ratios as the phenotypically wild flies. Since 18s 4- 28s rRNA genes have different locations from 5s RNA genes, it is presumed that Drosophila stocks can be constructed which have varying numbers of 18s 28s genes but same number of 5 s RNA genes. Bobbed genotypes offer such a possibility. They can be used to explore whether o r not the synthesis of 5s RNA is coordinated with 18s 4- 28s rRNA. Results presented here demonstrate that bobbed flies synthesize 5s RNA at a decreased rate but accumulate the same amount of 5s RNA as the wild-type (1972) has presented results which support our earlier obserflies. WIENMANN and RITOSSA1970) vations on 18s 28s rRNA synthesis in bobbed flies (MOHAN but are not in agreement with results on 5 s RNA synthesis presented in this study.
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MATERIALS A N D METHODS
Drosophila stocks: Drosophila melanoguster were raised on a cornmeal-yeast-agar medium. Wild-type Canton-S was obtained from the Universiiy of Pavia. uflbbl/BS Y , In(1) sc4L~8Rand car bb were from the Oak Ridge National Laboratory. X YL . YS bb was selected by DR. F. RITOSS~ from X Y L . y S (108-109 Parker) obtained from the University of Rome. These Drosophila stocks were kept in such genotypic combinations that the bobbed flies could be generated in one cross. The stocks were never kept in the bobbed condition as such because of the labile nature of this locus. So the bobbed flies were obtained prior to the experiments by crossing parents carrying both bb+ and bb- chroniosomes The flies which first emerged were not usually strongly bb and were discarded. The flies emerging later were strongly bb and were employed f o r the experiments. Four female genotypes werr employed for this study: Wild-type Canton-S bb+/bb+; car bb/In(l) S C ~ L , ~X RYL.Ys ; bb/wa bbl; and X YL.Ys bb/ln(1) S C ~ LI, n~(~Z. ) ~ c 4 ~ ~ ~ ~ is an X chromosome completely deficient in nucleolar orzanizer and rRNA genes (RITOSSA and SPIEGELMAN 1965). The symbol sc4 sc8 is used to denote this chromosome in the following text. For a complete drscription of Drosophila genetic no tation see LINDSLEY and GRELL(1968). Estimution of Ribosomal DNA: Labelled ribosomal RNA was obtained exclusively from the wild type larvae and purified by methylated albuniin kieselguhr (MAK) column. DNA from ATWOOD and SPIEGFLMAN 1966; ovaries and adult flies was prepared as described earlier (RITOSSA, M O H ~ 2nd N RITOSSA 1970). The method of GILLESPIE and SPIEGFLMAN (1965) for RNA/DNA hybridi7ation was followed. The most probable percentage of rDNA (average of at least four values) is given in parenthesis after each genotype in the text and tables. The details have already been presented (MOHAN and RITOSSA 1970). In uifro labelling of RNA in the ovaries: Seven-day-old females were mated to X / O sterile males to enhance rRNA synthesis (MOHAN1971b). The ovaries were dissected from females of variouq genotypes, washed in insect Ringer solution, and incubated for 30 and 60 min at 24O in Ringer solution containing 0.1 mCi of uridine-5-HS (24.4 Ci/m mole, Radiochemical Centre, Amersham, England). To obtain reproducible results the temperature was rigorously controlled.
R N A SYNTHESIS IN BOBBED MUTANTS
725
During incubation ovaries remained covered with paraffin oil. After incubation, ovaries were washed with Ringer solution to get rid of extracellular uridine and used for RNA extraction immediately. In vivo Zabelling ~f RNA: Radioactive medium containing 2.5 g cornmeal, 2.5 g sucrose, 1.0 g yeast, 20 ml water and 2 mCi 32phosphate was equally divided into three bottles. Seven-day-old mated flies were allowed to eat this food undisturbed for two hours a t 24". After this, flies were etherized and RNA was extracted immediately. Fractionation of R N A : Total RNA was extracted from ovaries and flies as described earlier (MOHANand RITOSSA 1970). Ethanol-precipitated RNA was dissolved in a small volume of 0.1 MNaCl and precipitated again by 2.0MNaCl at 4" overnight. The supernatant which contains low molecular weight RNA was dialyzed against water and concentrated by freezedrying. This RNA was subjected to polyacrylamide gel electrophoresis for 4 hr at 4 MA/gel as detailed by LOENING(1967). Gels Were scanned at 260 nm, frozen, sliced and counted (PERRY and KELLY1968). The high molecular weight RNA obtained after precipitation with 2.0 M NaCl was dissolved in 0.4 M NaCl and fractionated on a MAK column. Each fraction was precipitated with TCA, collected on nitrocellulose filter and the radioactivity determined in a liquid scintillation counter. Sucrose density gradients were done as described by SCHERERR and DARNELL (1962). Results were subjected to an analysis of variance and the mesn values of each bobbed genotype were compared with that of the wild type (bb+/bb+) by a t-test. RESULTS
To study the rate of RNA synthesis we have used the measure of specific activity, expressed as cpm/pg; the rationale behind this has already been discussed (MOHANand RITOSSA 1970). In order to use this as a parameter, one has to know the amount of various RNA's already present in the cell. To determine these values, total RNA was fractionated by several methods-i.e., MAK columns, polyacrylamide gel electrophoresis and sucrose density gradients. MAK column chromatography of two RNA preparations, obtained after precipitation with 2M NaC1, is shown in Figure 1. The optical density peak corresponds to combined 18s and 28s rRNA and elutes at about 0.8M NaCl. Figure 2 shows the results of polyacrylamide gel electrophoresis of a low molecular weight RNA preparation obtained from the supernatant of high salt precipitation. The 5 s RNA comes out as a sharp peak, while the 4s RNA fraction is broader. Drosophila 5s runs coincidentally with E . coli 5s RNA. A fraction slower than 5 s RNA and PERRY 1970). is also observed (TARTOF The percentage amount of various RNA's was calculated by using data obtained from different fractionation methods. Results of such an analysis in ovaries are presented in Table 1. RNA/DNA ratios were determined as described earlier (MOHANand RITOSSA 1970). Nearly 93% of the total RNA is represented by 18s 4- 28S, 5s and 5 s RNA. Approximately 1.6 molecules of 5s RNA are present for every molecule of 18s 28s RNA. Nearly eight molecules of 4s RNA are present in the cell for each 18s 28s RNA molecule. Analysis of nucleic acid content in adult flies of four genotypes is given in Table 2. Nearly 89% of the total RNA is accounted f o r by 18s 28S, 5s and 4s RNA. This figure is slightly lower than observed in ovaries. The molar ratio of 5s to 18s f 28s RNA in this case approaches unity. The number of 4s molecules is even greater than observed in ovaries. Except in the case of 5 s RNA which is slightly higher in ovaries of the X YL.Ysbb/wa bbl (0.120%) genotype as reflected by molar ratios, it is ob-
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J. MOHAN
FRACTION NUMBER
FIGURE 1.-MAK column chromatography of two RNA preparations obtained after precipitation with 2 M NaCI. Approximate;ly 200 pg of RNA was loaded. High molecular weight RNA's were eluted by a linear gradient of NaCl from 0.5 M-I .2 M with a total volume of 50 ml. Two-ml fractions were collected. The optical density peak corresponds to 18s f 28s rRNA and eluted at about 0.8M NaCl and the radioactivity peak to rRNA precursor 38s and eluted at about 0.9 M NaCI. Nearly 85%-90% of the radioactivity was recovered. A. In vitro RNA synthesis after incubating ovaries for 60 minutes in 3H-uridine. .---e O.D. at260nmO-0 3HCPMbb+/bb+ (0.360). A-A 3H CPM X Y L . Y S bb/sc4 S C ~(0.090). B. I n vivo RNA synthesis after feeding flies on 3Zphosphatemedium for 2 hours. .----e O.D. at 260 nm. 0-0 32PCPM bb+/bb+ (0.360). A-A 32P CPM X YL.YS bb/wa bbz (0.120).
vious from Tables 1 and 2 that no significant differences exist in ovaries and adult flies, between genotypes carrying from nearly 0.085% to 0.360% rDNA. There is a greater amount of RNA per unit DNA in ovaries as compared to adult flies. The ovaries also contain more molecules of 5s RNA. We did not see a reduced amount of 5s RNA in the car bb (bb6)/scAssc8(0.085%) genotype, as reported by WEINMANN (1972). It was demonstrated earlier that the rate of 18s 28s synthesis is reduced in Drosophila melanogaster flies exhibiting a bobbed phenotype (MOHANand RITOSSA1970). This observation has been subsequently confirmed by others (WEINMANN 1972; KRIDERand PLAUT 1972). This is due to the fact that these flies contain subnormal amounts of rDNA. However, such flies maintain a normal rate of 4s RNA synthesis. So to avoid differences in pool size, food composition, amounts of food eaten and other experimental variables, the results could
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727
R N A SYNTHESIS IN BOBBED MUTANTS
Department of Genetics, Haryana Agricultural University, Hissar-125004,
India
Manuscript received February 3, 1975 Revised copy received June 15, 1975 ABSTRACT
Ribosomes contain one molecule each of 5S, 18s and 28s RNA. In Drosophila melanogaster although the genes for 18s 28s are physically separated from the 5s RNA genes, the multiplicity of various ribosomal RNA genes is roughly the same. Thus a coordinate synthesis of these three molecules might seem feasible. This problem has been approached by determining the molar ratios of various R N A s i n ovaries and in adult flies. In ovaries there is a slight excess of 5s RNA molecules over other rRNA's, but in adult flies no such differences exist. Bobbed mutants also have the same molar ratios as wild-type flies. Results on 5s RNA synthesis in both in vitro and in vivo studies show that it is reduced in coordination with 18s 28s rRNA in the bobbed mutants of Drosophila melanogaster. Various possibilities are discussed in considering the implications of these results.
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+
RIBOSOMES contain one molecule each of three distinct types of ribonucleic acid (rRNA) conventionally known by their sedimentation values as 28S, 18s and 5s in higher organisms. Genes for 28s and 18s rRNA are localized a t CHIPCHASE and SPIERS1971). the sites of the nucleolar organizers (BIRNSTIEL, In Drosophila melanogaster 5 s RNA genes are clustered away from nucleolar and STEFFENSEN organizer at the 56F locus of the second chromosome (WIMBER STEFFENSEN and HUGHES 1973) and their number is in near 1970; PRENSKY, parity to 18s and 28s rRNA genes (TARTOF and PERRY 1970; QUINCEY1971). This differs sharply from the situation observed in other eukaryotes where genes and ATTARDI for 5s RNA are spread over several chromosomes (ALONI,HALTEN 1971; PARDUE, BROWNand BIRNSTIEL1973; STEFFENSEN and DUFFEY1974) and there is an excess of these genes over 18s and 28s rRNA genes (BROWN and WEBER 1968; QUINCEYand WILSON 1969; AMALDIand BUONGIORNO-NARDELLI 1971; BIRNSTIEL,SELLSand PURDOM 1972; BROWNand SUGIMOTO 1973). In most cells three types of rRNA's are present in equimolar amounts (DENIS, WEGNEZ and MAIRY1973). However, 5s RNA synthesis is not always coordinated (ABE and YAMANA 1971). During early oogenesis in Xenopus, there is 1971) . I n mamnon-coordinated synthesis and accumulation of 5s RNA (FORD malian cells, 5s RNA synthesis continues in the absence of 18s and 28s synthesis Genetics 81: 723-738 Decembei, 1975
724
J. MOHAN
(PERRY and KELLY1968) and there is an excess of 5s RNA synthesis (LIEBOWEINBERG and PENMAN 1973). In the present study 5s RNA synthesis and its rela tionship to 18s -I-28s rRNA in Drosophila melanogaster has been studied. It is possible by genetic manipulation to alter the number of 18s 28s rRNA genes in this organism (RITOSSA and SPIEGELMAN 1965; RITOSSA,ATWOOD and SPIEGELMAN 1966). Drosophila geno-
WITZ.
+
types containing less than 130 such genes manifest a bobbed phenotype characterized by slow development, smaIl bristles and etching of the abdominal tergites. We have shown (MOHAN and RITOSSA1970) that the rate of 18s -I-28s rRNA synthesis is reduced in bobbed flies, but they have the same RNA/DNA ratios as the phenotypically wild flies. Since 18s 4- 28s rRNA genes have different locations from 5s RNA genes, it is presumed that Drosophila stocks can be constructed which have varying numbers of 18s 28s genes but same number of 5 s RNA genes. Bobbed genotypes offer such a possibility. They can be used to explore whether o r not the synthesis of 5s RNA is coordinated with 18s 4- 28s rRNA. Results presented here demonstrate that bobbed flies synthesize 5s RNA at a decreased rate but accumulate the same amount of 5s RNA as the wild-type (1972) has presented results which support our earlier obserflies. WIENMANN and RITOSSA1970) vations on 18s 28s rRNA synthesis in bobbed flies (MOHAN but are not in agreement with results on 5 s RNA synthesis presented in this study.
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MATERIALS A N D METHODS
Drosophila stocks: Drosophila melanoguster were raised on a cornmeal-yeast-agar medium. Wild-type Canton-S was obtained from the Universiiy of Pavia. uflbbl/BS Y , In(1) sc4L~8Rand car bb were from the Oak Ridge National Laboratory. X YL . YS bb was selected by DR. F. RITOSS~ from X Y L . y S (108-109 Parker) obtained from the University of Rome. These Drosophila stocks were kept in such genotypic combinations that the bobbed flies could be generated in one cross. The stocks were never kept in the bobbed condition as such because of the labile nature of this locus. So the bobbed flies were obtained prior to the experiments by crossing parents carrying both bb+ and bb- chroniosomes The flies which first emerged were not usually strongly bb and were discarded. The flies emerging later were strongly bb and were employed f o r the experiments. Four female genotypes werr employed for this study: Wild-type Canton-S bb+/bb+; car bb/In(l) S C ~ L , ~X RYL.Ys ; bb/wa bbl; and X YL.Ys bb/ln(1) S C ~ LI, n~(~Z. ) ~ c 4 ~ ~ ~ ~ is an X chromosome completely deficient in nucleolar orzanizer and rRNA genes (RITOSSA and SPIEGELMAN 1965). The symbol sc4 sc8 is used to denote this chromosome in the following text. For a complete drscription of Drosophila genetic no tation see LINDSLEY and GRELL(1968). Estimution of Ribosomal DNA: Labelled ribosomal RNA was obtained exclusively from the wild type larvae and purified by methylated albuniin kieselguhr (MAK) column. DNA from ATWOOD and SPIEGFLMAN 1966; ovaries and adult flies was prepared as described earlier (RITOSSA, M O H ~ 2nd N RITOSSA 1970). The method of GILLESPIE and SPIEGFLMAN (1965) for RNA/DNA hybridi7ation was followed. The most probable percentage of rDNA (average of at least four values) is given in parenthesis after each genotype in the text and tables. The details have already been presented (MOHAN and RITOSSA 1970). In uifro labelling of RNA in the ovaries: Seven-day-old females were mated to X / O sterile males to enhance rRNA synthesis (MOHAN1971b). The ovaries were dissected from females of variouq genotypes, washed in insect Ringer solution, and incubated for 30 and 60 min at 24O in Ringer solution containing 0.1 mCi of uridine-5-HS (24.4 Ci/m mole, Radiochemical Centre, Amersham, England). To obtain reproducible results the temperature was rigorously controlled.
R N A SYNTHESIS IN BOBBED MUTANTS
725
During incubation ovaries remained covered with paraffin oil. After incubation, ovaries were washed with Ringer solution to get rid of extracellular uridine and used for RNA extraction immediately. In vivo Zabelling ~f RNA: Radioactive medium containing 2.5 g cornmeal, 2.5 g sucrose, 1.0 g yeast, 20 ml water and 2 mCi 32phosphate was equally divided into three bottles. Seven-day-old mated flies were allowed to eat this food undisturbed for two hours a t 24". After this, flies were etherized and RNA was extracted immediately. Fractionation of R N A : Total RNA was extracted from ovaries and flies as described earlier (MOHANand RITOSSA 1970). Ethanol-precipitated RNA was dissolved in a small volume of 0.1 MNaCl and precipitated again by 2.0MNaCl at 4" overnight. The supernatant which contains low molecular weight RNA was dialyzed against water and concentrated by freezedrying. This RNA was subjected to polyacrylamide gel electrophoresis for 4 hr at 4 MA/gel as detailed by LOENING(1967). Gels Were scanned at 260 nm, frozen, sliced and counted (PERRY and KELLY1968). The high molecular weight RNA obtained after precipitation with 2.0 M NaCl was dissolved in 0.4 M NaCl and fractionated on a MAK column. Each fraction was precipitated with TCA, collected on nitrocellulose filter and the radioactivity determined in a liquid scintillation counter. Sucrose density gradients were done as described by SCHERERR and DARNELL (1962). Results were subjected to an analysis of variance and the mesn values of each bobbed genotype were compared with that of the wild type (bb+/bb+) by a t-test. RESULTS
To study the rate of RNA synthesis we have used the measure of specific activity, expressed as cpm/pg; the rationale behind this has already been discussed (MOHANand RITOSSA 1970). In order to use this as a parameter, one has to know the amount of various RNA's already present in the cell. To determine these values, total RNA was fractionated by several methods-i.e., MAK columns, polyacrylamide gel electrophoresis and sucrose density gradients. MAK column chromatography of two RNA preparations, obtained after precipitation with 2M NaC1, is shown in Figure 1. The optical density peak corresponds to combined 18s and 28s rRNA and elutes at about 0.8M NaCl. Figure 2 shows the results of polyacrylamide gel electrophoresis of a low molecular weight RNA preparation obtained from the supernatant of high salt precipitation. The 5 s RNA comes out as a sharp peak, while the 4s RNA fraction is broader. Drosophila 5s runs coincidentally with E . coli 5s RNA. A fraction slower than 5 s RNA and PERRY 1970). is also observed (TARTOF The percentage amount of various RNA's was calculated by using data obtained from different fractionation methods. Results of such an analysis in ovaries are presented in Table 1. RNA/DNA ratios were determined as described earlier (MOHANand RITOSSA 1970). Nearly 93% of the total RNA is represented by 18s 4- 28S, 5s and 5 s RNA. Approximately 1.6 molecules of 5s RNA are present for every molecule of 18s 28s RNA. Nearly eight molecules of 4s RNA are present in the cell for each 18s 28s RNA molecule. Analysis of nucleic acid content in adult flies of four genotypes is given in Table 2. Nearly 89% of the total RNA is accounted f o r by 18s 28S, 5s and 4s RNA. This figure is slightly lower than observed in ovaries. The molar ratio of 5s to 18s f 28s RNA in this case approaches unity. The number of 4s molecules is even greater than observed in ovaries. Except in the case of 5 s RNA which is slightly higher in ovaries of the X YL.Ysbb/wa bbl (0.120%) genotype as reflected by molar ratios, it is ob-
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J. MOHAN
FRACTION NUMBER
FIGURE 1.-MAK column chromatography of two RNA preparations obtained after precipitation with 2 M NaCI. Approximate;ly 200 pg of RNA was loaded. High molecular weight RNA's were eluted by a linear gradient of NaCl from 0.5 M-I .2 M with a total volume of 50 ml. Two-ml fractions were collected. The optical density peak corresponds to 18s f 28s rRNA and eluted at about 0.8M NaCl and the radioactivity peak to rRNA precursor 38s and eluted at about 0.9 M NaCI. Nearly 85%-90% of the radioactivity was recovered. A. In vitro RNA synthesis after incubating ovaries for 60 minutes in 3H-uridine. .---e O.D. at260nmO-0 3HCPMbb+/bb+ (0.360). A-A 3H CPM X Y L . Y S bb/sc4 S C ~(0.090). B. I n vivo RNA synthesis after feeding flies on 3Zphosphatemedium for 2 hours. .----e O.D. at 260 nm. 0-0 32PCPM bb+/bb+ (0.360). A-A 32P CPM X YL.YS bb/wa bbz (0.120).
vious from Tables 1 and 2 that no significant differences exist in ovaries and adult flies, between genotypes carrying from nearly 0.085% to 0.360% rDNA. There is a greater amount of RNA per unit DNA in ovaries as compared to adult flies. The ovaries also contain more molecules of 5s RNA. We did not see a reduced amount of 5s RNA in the car bb (bb6)/scAssc8(0.085%) genotype, as reported by WEINMANN (1972). It was demonstrated earlier that the rate of 18s 28s synthesis is reduced in Drosophila melanogaster flies exhibiting a bobbed phenotype (MOHANand RITOSSA1970). This observation has been subsequently confirmed by others (WEINMANN 1972; KRIDERand PLAUT 1972). This is due to the fact that these flies contain subnormal amounts of rDNA. However, such flies maintain a normal rate of 4s RNA synthesis. So to avoid differences in pool size, food composition, amounts of food eaten and other experimental variables, the results could
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727
R N A SYNTHESIS IN BOBBED MUTANTS
