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Regulation in rDNA-deficient Drosophila melanogaster
Cell, Vol . 4, 275-280, March 1975, Copyright©1975 by MIT
Regulation in rDNA-Deficient Drosophila melanogaster
A. W . Shermoen* and B . I . Kiefer Department of Biology Wesleyan University Middletown, Connecticut 06457
Summary Bobbed mutants of Drosophila melanogaster were used to determine whether there is any functional compensation for deficiency in rDNA content . The rate of rRNA accumulation was measured in the testes of five bb mutants of different phenotypic severity and in a wild type strain . The different rates of rRNA accumulation were compared to the phenotype (macroscutellar bristle length) and found to have a direct correlation (as in Weinman, 1972) . However, there was not a direct relationship between the rate of rRNA accumulation and rDNA content . It is concluded that there is regulation of rRNA accumulation in some mutants, but that the regulation cannot be considered to be a compensation for the lack of rDNA . These results are discussed relative to other observations on the regulation of RNA synthesis in Drosophila . Introduction Ribosomal RNA has been the subject of many studies on gene activity in eucaryotes because of its accessibility as a primary gene product . Despite the mass of published work on the synthesis and processing of rRNA, we have yet to come to an understanding of the control of its transcription . The analysis of mutants affecting the process of rRNA synthesis is an especially attractive way to investigate the genetic activity of rDNA . Drosophila melanogaster is a choice organism for this approach because of the existence of a large number of mutants which are at least partial deletions of rDNA (that is, bobbed or bb) (Ritossa, Atwood, and Spiegelman, 1966a) . rDNA has been found to be localized to NO regions, which in this species are two in number and reside on the X and on the Y chromosomes (Ritossa and Spiegelman, 1965) . The bb mutants are recognized most easily by their relatively short macroscutellar bristles . They also generally have a thinner cuticle and longer period of development than wild type . While a study of rRNA gene activity may constitute a special case of regulation, there are structural and functional correlations with other genes . Other redundant genes which are known are 5S RNA, *Present address : Department of Zoology, University of California, Berkeley, California 94720 .
tRNA, histone genes, and some genes involved in spermatogenesis in Drosophila (Brown and Weber, 1968 ; Ritossa et al ., 1966b ; Kedes and Birnstiel, 1971 ; and Hennig, 1968) . A further similarity between rDNA and other genes is indicated by evidence that many immediate gene products may be transcribed as a large precursor RNA which is then processed to the smaller product found in the cytoplasm (Melli and Pemberton, 1970 ; Lindberg and Darnell, 1970 ; Maden, 1971 ; Firtel and Lodish, 1973) . A study of rRNA synthesis therefore may provide information concerning both transcriptional and posttranscriptional control mechanisms of gene expression . Two ways in which rRNA synthesis is regulated in Drosophila have already been described . Both mechanisms of regulation involve the increase in the amount of template present for transcription . "rDNA compensation," a single generation increase in rDNA content, is restricted to somatic cells and occurs when rDNA on the X chromosome is opposite another chromosome deficient in rDNA, such as X/O or X/Ybb - males and X/X"o- females (Tartof, 1971, 1973) . [Genetic nomenclature for rDNA mutants in D . melanogaster : bb and bbds = partial deletions ; - bb, bb-, and NO- = virtually complete deletions ; Ybb- has about 30 rRNA genes (Tartof, 1973) .] "Magnification", on the other hand, occurs in germ cells . When a bb chromosome is maintained over several generations opposite another bb chromosome, such as Xbb / Ybb there is a gradual increase of rDNA so that Xbb reverts to Xbb+ (Ritossa, 1968 ; Tartof, 1974) . Previous work measuring the rate of rRNA synthesis in ovaries relative to the rDNA content of the fly was interpreted to show an unmediated use of the rDNA in bb mutants (Mohan and Ritossa, 1970 ; Weinman, 1972) . These authors concluded that there was a constant rate of rRNA synthesis/gene for all flies which contained up to the haploid amount of rDNA . Flies containing a greater than haploid rDNA content did not have an increase in rRNA synthesis (Mohan and Ritossa, 1970 ; Krider and Plaut, 1972) . These investigations would all seem to indicate that in Drosophila, a primary method of regulating rRNA production lies at the level of altering the template content of the cell . There are, of course, other possibilities of control . The work reported here involved the use of bobbed flies whose phenotype varied over a wide spectrum . Two questions were of primary concern : -Is there regulation of rRNA synthesis in rDNA-deficient mutants? -If such regulation exists, is it correlated with the amount of rDNA present?
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Our experiments show that a model of rRNA regulation in Drosophila melanogaster based solely upon the amount of ribosomal genetic material present is too simplified .
by others (Tartof, 1971 ; Williamson, Procunier, and Church, 1973) and was roughly four times the rDNA content of the bb genotypes, which clustered around 100 genes . ybb Su Var 5 and Y bb- chromosomes have previously been measured and shown to be equivalent in rDNA content (Malva et al ., 1972) . The first question asked was whether the bristle length is directly representative of the rDNA content. As Figure 1 shows, the answer is no . It is apparent that the bristle length-rDNA content relationship does not hold for wild type relative to bb, and it is also evident that no such relationship exists among the bb mutants . This is the first indication that there may be a regulation of rDNA cistrons in bb flies . Next the rates of rRNA accumulation were measured for the testes of each of the genotypes, and are represented as specific activities . Use of the mass (µg) of rRNA as standardization for the dpm accumulation by 18S and 28S is legitimate since measurement of the RNA/testis revealed that the most severe bb (car bb/Ybb - ) contained only 20% less RNA/organ than did wild type (0 .84 µg/testis and 1 .05 µg/testis, respectively) . The differences in these bb specific activities therefore would be somewhat higher than the real rate of accumulation, but the difference is insignificant relative to the degree of change which has been measured . Another way in which the specific activities are verified as being truly representative of the rates of rRNA accumulation (and probably synthesis) is that the amount of accumulation of radioactivity as tRNA (as
Results Measurements of the posterior macroscutellar bristles are shown in order of their increasing length in Table 1 . These genotypes were selected because their bb phenotype varied in severity . One could therefore determine not only whether there is regulation of rRNA synthesis, but also whether that regulation is related to the rDNA content . The rDNA content of each genotype was measured by saturation hybridization of 3 H-rRNA to DNA isolated from adult males (Table 2) . The wildtype rDNA content is approximately that reported Table 1 . Macroscutellar Bristle Lengths
(µ)
Genotype
Bristle Length
y w bbd•/ Ybb -
282
Standard Deviation 33
car bb/Ybb-
285
16
gt bb'T/Ybb -
326
8
y w bbd•/ Y - bb
345
24
y w bbd•/ Ybb Su Var 5
391
27
Samarkand+ iso
365
19
The posterior dorsal thoracic (macroscutellar) bristles were measured by an ocular micrometer in a dissecting microscope . The males measured were from the cultures used to obtain DNA for the hybridization experiments .
Table 2 . 3H-rRNA-DNA Saturation Hybridization Levels Genotype
rRNA hybridization x 100 DNA
Standard Deviation
Gene Number
y w bbd•/ Ybb -
0 .0889
0 .0026
101
car bb/Ybb-
0 .0783
0 .0118
89
gt bbl /Ybb-
0 .0805
0 .0041
92
y w bbd•/ Y - bb
0 .1076
0 .0171
123
y w bbd•/ Ybb Su Var 5
0 .0915
0 .0166
104
Samarkand+ iso
0 .3555
0 .0387
405
Determination of rDNA content was made by rRNA-DNA hybridization (Gillespie and Gillespie, 1971) . A minimum of six determinations were made for each saturation point . Blank filters and filters containing E . coli DNA were used to monitor background radioactivity . The calculation of rRNA gene number was made in the following way : rRNA specific activity 75,700 cpm/µg
DNA/filter 5 .96 µg
rRNA cpm filter 414 cpm
414 cpm = 0 .00547 µg rRNA 75,700 cpm/µg 0 .00547 µg rRNA x 100 = 0 .0918% hybridization 5 .96 µg DNA 0 .0918% x 2 = 0 .1836% rDNA (2 .4 x 10 1 1 daltons EDNA) (1 .836 x 10-3 rDNA) _ 104 .8 genes 4 .2 X 106 daltons rDNA
diploid DNA 2 .4 x 10 11 daltons
18S + 28S 2 .1 x 106 daltons
Regulation in rDNA-Deficient D . melanogaster 277
seen by 10% polyacrylamide gel electrophoresis) is similar for genotypes which vary widely in rDNA (car bb /Y bb- , Sam++ iso) (Mohan and Ritossa, 1970 ; Clark, 1975) . These results indicate both that the precursor pools have very similar specific activities and that the same amount of DNA is represented in all of the tissues tested . Furthermore, possible differences in time of pool equilibration were compensated for by subtracting the dpm accumulated in the first hour of incorporation from the dpm incorporated after 3 hr . Figure 2 is a plot of the rate of radioactive rRNA accumulation compared with the bristle length and shows a direct relationship between the two parameters . This is the same result as Weinmann (1972), who used different conditions including an isogenic background for testing single X chromosomes . However, the substantive relationship (for the question of rate regulation) is shown in the plot of the rate of rRNA synthesis relative to the rDNA content (Figure 3) . The rate of rRNA synthesis is different for wild type than for the bb flies, and even within the series of bb mutants there is not a clear direct relationship. The rate problem is put in clearer perspective if one divides the rate of accumulation by the gene number and relates the
rate of accumulation/gene to the rDNA content . If there is a direct relationship of the rate of rRNA accumulation to rRNA gene number, this graph will have a straight line parallel to the abscissa . This is clearly not the case (Figure 4) . Discussion In agreement with Weinmann (1972), the data shown here (Figure 2) demonstrate a direct relationship between the rate of radioactive rRNA accumulation and bristle length . This agreement in results comes despite the care taken by Weinmann to use a completely isogenic background changing only the tested chromosome . The similarity is striking for two reasons . First, Weinmann used two chromosomes that were also used in this study, gt bbl 1 and car bb ; if the ratio of relative rates for each genotype ( 9a b,,°,) of Weinmann's data (Table 3, Weinmann, 1972) is compared with the data here they correspond well within experimental error, 0 .49 and 0 .52, respectively . In addition, the correlation shown here comes from the rate of accumulation as measured in spermatogonia and primary spermatocytes, whereas Weinmann's data uses the rate
500
y w bbd,
50
450
45
Sam* ISO
400
40
35
350
L Q)
30
300
M
O X E
25 250
Z
a) c
20 200
E a v
y w bbds 150
y w bbds
Ybb
Ybb 100
50
15 _I
Y -bb 10
I
ST i
y w bb ds
gt bb
car bb Y bb -
Ybb-
Ybb
5
Su Var 5 i
240 I
260
I
300
I
I
I
340
380
I
A
I
280
L
I
320
i
L
360
1
1
400
Bristle length ()u)
420
Bristle length (,u) Figure 1 . Bristle Length Relative to rRNA Gene Number The macroscutellar bristle lengths (Table 1) are graphed relative to the rRNA gene number (Table 2) for their respective genotype . The bb genotypes do not have a linear relationship .
Figure 2 . A Plot of Bristle Length Relative to rRNA Accumulation The relationship of macroscutellar bristle length is shown relative to the rate of radioactive rRNA accumulation in the testes for each genotype (see Experimental Procedures for details) . Isotope incorporation is standardized to the rRNA content of each genotype (see Results section for a discussion of this use of specific activity) .
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of accumulation in whole female D melanogaster . Both of these sets of data are related to the product of a third cell type, the trichogen cells that produce the bristles . These tissue differences imply that if regulation of rRNA synthesis is found it will be due to something other than a cytoplasmic feedback mechanism, since one would expect to find a variety of physiological conditions in these disparate cell types. Figures 3 and 4 show there is regulation of rRNA accumulation, and that this regulation of the efficiency of expression of the rRNA genes is not a function of the number of rRNA genes present . These data contradict the interpretation of their data by Mohan and Ritossa (1970), who maintained that there is a constant rate of rRNA synthesis through the "haploid" rDNA content . Miller and Knowland (1970) found a somewhat lower than wild type rate of rRNA synthesis/gene in a partial deletion of rDNA in Xenopus laevis, again indicating a correlation between the number of rRNA genes and accumulation of rRNA when rDNA is below some threshold level . Although our data indicate that some form of regulation is occurring, it is not yet possible to clearly distinguish between transcriptional and posttranscriptional control in these studies . Some restrictive regulation of rRNA synthesis in Drosophila has previously been discovered in addition to work by
Mohan and Ritossa (1970) . Krider and Plaut (1972) found that the rate of incorporation of 3 H-U by salivary gland nucleoli is identical for genotypes of one to four intact NOR and that, seemingly, no more than two NOs are active at any one time . Second, Meyer and Hennig (1974) counted active rRNA cistrons from EM spreads and concluded that only about half of the NOR are active in primary spermatocytes of Drosophila hydei . They also think that it is likely that not all of those cistrons which eventually function are active in early primary spermatocytes . Finally, Baker (1971) and Puckett and Snyder (1974) showed that there are some sct inversions (that is, an inversion which includes one break point adjacent and proximal to the NOR) with a full complement of rDNA which is subject to position-effect restriction . [Position-effect variegation is considered to be a restriction of the transcriptive activity which occurs in some cases of translocations and inversion of euchromatin to heterochromatin (Baker, 1968) .] Two interesting observations relative to positioneffect variegation (PEV) and its possible relationship to the rDNA region may be mentioned here . Many studies have demonstrated that PEV can be suppressed by the addition of heterochromatin . Brosseau (1964) demonstrated that there were two regions of the heterochromatic Y chromosome which were particularly effective in overcoming PEV 500
y w bbd,
50
. 1
i ,~i y w bbds
Y bb Su Var 5
Ybb Su Var 5 450
Sam' ISO 45
F-~1
40
400
y w bbds y -bb
350 35
y w bbds y -bb
a, C
fr1
O
p
X
4H gt bb" y b b-
0+
Z
gt bb"
0+ Iw
?
300
30
y bb -
25
250
E C1. 0
E . 0 -0 20
200
150 15
car bb ybb -
car bb "JV Ybb-
y w bb Y bb
d'
y w bbds ybb I+-1
y Sam + ISO
100
10 50 5
0 .1 0 .1
0.2 rRNA / DNA x
0.3
0 .2 rRNA /DNA x
0.3
0 .4
100
0 .4
100
Figure 3 . A Plot of rRNA Gene Number to rRNA Accumulation The saturation level of rRNA hybridization is graphed against 3H rRNA accumulation .
Figure 4 . A Plot of rRNA Gene Number to rRNA Accumulation/ Gene This graph demonstrates the average rate of utilization of rRNA genes for each genotype . Two bb mutants and wild type have the same average rates of utilization .
Regulation in rDNA-Deficient D . melanogaster 2 79
of an X chromosome locus . These two regions were located near the fertility loci, kl-1 and ks-1 . ks-1 is adjacent and distal to the NOR on the Y chromosome . Second, it is known that rearrangements of the It locus, located in heterochromatin of chromosome 2, also express a PEV . However, this heterochromatic rearrangement [R(/t+)] responded oppositely to added Y chromosomes as compared to a euchromatic rearrangement [R(w+)] . That is, It+ was most active (normal) in the X/O condition, and progressively decreased its activity with addition of heterochromatin (Baker and Rein, 1962) . Hence, those heterochromatic regions which are so successful in restoring gene activity to R(euchromatin) seem to restrict the activity of at least one example of R(heterochromatin) . These various examples of transcriptive regulation in Drosophila melanogaster suggest several conclusions in light of the data presented in this report . (a) Because there are examples of transcriptive regulation and a lack of natural examples for the regulation of RNA processing, it is simplest to interpret the increased rate of rRNA accumulation for three genotypes, y w bbds/Y - bb, gt bbl l/Ybb-, and y w bbds/Ybb Su Var 5 as an increase in the average rate of rRNA synthesis/gene . Since the change in the rate of rRNA synthesis is not correlated with the rDNA content of the fly, the explanation proposed to account for the observed rate differences is that there is a region external (and adjacent) to the rDNA which has affected the utilization of the rRNA gene . This suggestion also has been made by Procunier and Williamson (1974) from work on temperature sensitive bb mutants . Furthermore, the normal (wild type) condition of rDNA activity is under restriction, and it is only in some mutants in which this restricted transcription has been alleviated . (b) Second, those chromosomes which display more efficient use of their rDNA may have rearrangements within the heterochromatin which either separate the rDNA from those regions which suppress euchromatin PEV or destroy, at least in part, such regions . One prediction from the first proposal would be that since an X chromosome bb mutant did show an increase in rRNA synthesis/gene (gt bb"), and Baker (1971) found some sct chromosomes in which rRNA synthesis is depressed (thereby eliminating the proximal side of the NOR from consideration), we would postulate that some sc 4 inversions (breakpoints distal to the NOR) would show an increase in rRNA synthesis/gene . A prediction from the second proposal would be that Ybb chromosomes which have an increased
rate of rRNA synthesis/gene should have the same or decreased ability to suppress PEV . These and more direct experiments relative to the nature of the regulatory mechanism(s) involved are currently being tested . Experimental Procedures Drosophila Stocks A list of the crosses and the progeny used in these experiments is provided below . All genotypes were obtained from I . I . Oster at Bowling Green University . Cross
F, Used
wm4 /Ybb- x ywbbds/ywbbd ,
ywbbds/Ybb -
wm4/Ybb- x car bb/car bb
car bb/Ybb -
wm•/ Ybb - X gt bb""/c1 B
gt bbl I / Ybb-
w sn bb/Y-bb x y w bbd'/y w bbd ,
y w bbd,/Y-bb
wm4/Ybb Su Var 5 x y w bbd,/y w bbds
y w bbds/Ybb SU Var 5
Samarkand isogenic Wild type
Sam+ iso
DNA Extraction DNA was extracted by modification of a method developed by L . Weber (personal communication) . At least 5 g of adult males of any one genotype were ground to a fine powder with a dry icecooled mortar and pestle . The powdered flies were slowly stirred into 9 vol of room temperature NETM buffer (0 .1 M NaCl, 0 .01 M EDTA, 0 .03 M Tris, 0 .01 M 8-mercaptoethanol, pH 8 .0) with Triton X-100 added to 0 .5% . The mixture was then homogenized twice with a Dounce glass homogenizer and "B" pestle . Cuticle and large tissue masses were sedimented with a hand centrifuge, and then the supernatant was centrifuged at 5000 rpm for 10 min in the SS-34 Sorvall rotor . The nuclear pellet was resuspended in 5 vol of NETM buffer and homogenized as before . The nuclei were sedimented again and resuspended in T E buffer (0 .1 M Tris, 0 .1 M EDTA, pH 8 .4). SDS was added to a concentration of 2%, and then the solution was shaken gently for 20 min . 5 M NaCl 04 was added to a final concentration of 1 M NaCl 04 . The mixture was shaken gently for 20 min . The DNA was then extracted once with 1 vol buffer saturated neutralized phenol, and twice with 1 vol of chloroform/isoamyl alcohol (24 :1, v/v) . DNA was precipitated with 2 vol 95% ethanol and resuspended in 1 x SSC. The DNA solution was treated with 100 µg/ml pancreatic ribonuclease X11-A (Sigma), 20 units/ ml T1 ribonuclease (Worthington), and 200 µg/ml a-amylase (Sigma) for 2 hr . Pronase B (Calbiochem), preincubated for 4 hr at 37°C, was added to 50 pg/ml and incubated for an additional hour . DNA was then extracted 3 times or more with equal volumes of chloroform/isoamyl alcohol until the 260/280 ratio was 1 .95 or greater. The DNA was precipitated with 2 vol of 95% ethanol . DNA was resuspended in 0 .1 x SSC and centrifuged at 42,000 rpm in an SW-50 .1 Spinco rotor for 30 min in order to sediment any remaining polysaccharides. All DNA solutions had a 260/230 ? 2 . Labeling and Extraction of rRNA for Hybridization (Modification from Ritossa et al ., 1966a.) Samarkand strain embryos were collected from a yeast plate that had been in a population cage for 3-4 hr . Two-tenths g of embryos were placed on 21 ml of medium in a crystallizing dish (10 cm diameter x 5 cm height) . The medium contained 16 ml of mixture A [100 ml distilled H 2O, 10 g Instant Drosophila Medium (Carolina Biological), 2 g Fleischmans Yeast, 0 .8 g Bacto agar] and 5 ml uridine-5- ; H (1 me/ml ; 28 curies/mM ; New England Nuclear) . The dishes were covered with cheesecloth and kept at high humidity and 25°C . After 1 week the larvae were collected on nylon stocking (Kirsten) and washed with distilled water . The rRNA was extracted according
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to Tartof and Perry (1970), and the 28S and 18S rRNA collected individually from 5-20% sucrose gradients . Hybridization was done using 28S and 18S in 2 :1 ratio by UV absorbance . The rRNA specific activity was 75,700 cpm/µg . Hybridization Procedures DNA was denatured by alkali or by boiling for 10 min in 0 .1 x SSC, then adsorbed to 25 mm nitrocellulose membrane filters (Schleicher and Schuell, type B-6) according to the methods of Gillespie and Spiegelman (1965) . The amount of DNA adsorbed to the filters was determined by measuring the A260 prior to filtration, and then the A 260 of the filtrate, and by the retained radioactivity of radioactively labeled E . coli DNA and S . purpuratus DNA (gift of Dr . C. Alfageme) . In most cases "minifilters" (5 mm diameter) punched from the large filters were used in hybridization . Filters containing radioactive DNA were tested for an even distribution of DNA over the surface of the filter . Minifilters contained from 5-10 µg/filter . The hybridization and posthybridization process methods used were esentially those of Gillespie and Gillespie (1971), with the modification that the incubation medium was 50% formamide (Eastman Kodak Co.)-2 x SSC, (pH 7 .0), and 0 .2-0 .5% SDS . Radioactivity retained as hybrid was counted in a N/C liquid scintillation spectrophotometer for a time that resulted in no more than a 1 % counting error .
Received November 11, 1974 ; revised December 23, 1974 References Baker, W . K . (1968) . Advan . Genet. 14, 133-169 . Baker, W . K . (1971) . Proc . Nat. Acad . Sci . USA 68, 2472-2476 . Baker, W . K ., and Rein, A. (1962) . Genetics 47, 1399-1407. Brosseau, G . E. (1964) . Genetics 50, 237 . Brown, D . D ., and Weber, C . S . (1968) . J . Mol. Biol . 34, 661-680 . Carlson, E . A., and Oster, I . I . (1962). Genetics 47, 561-576 . Clark, S . H . (1975) . Ph .D . Thesis, Wesleyan University, Middletown, Connecticut . Firtel, R . A ., and Lodish, H . F . (1973) . J . Mol . Biol . 79, 295-314 . Gillespie, S ., and Gillespie, D . (1971) . Biochem . J . 125, 481-487 . Gillespie, D ., and Spiegelman, S . (1965) . J . Mol . Biol. 12, 829-842 . Hennig, W . (1968) . J . Mol . Biol . 38, 227-239 . Kedes, L. H ., and Birnstiel, M . L . (1971) . Nature New Biol . 230, 165-169 . Krider, H ., and Plaut, W . (1972) . J . Cell Sci . 11, 675-687 . Lindberg, U ., and Darnell, J . (1970) . Proc. Nat . Acad. Sci . USA 65, 1089-1096. Loening, U . E . (1969) . Biochem . J . 113, 131-138 .
Measurement of rRNA Synthesis Incorporation of radioactive isotope : 2-3 H-adenosine (17 .7 curies/ mM, New England Nuclear) was lyopholized and resuspended in sterile Drosophila Ringers at a concentration of 20 me/ml . Approximately 0 .25 µl (5 µc) was injected into the abdomen of each lightly etherized fly with a hand-pulled glass needle (Carlson and Oster, 1962) . After 1 or 3 hr of incorporation, the flies (30 flies/group) were frozen in a vial on dry ice . The testes were dissected into ice-cold Drosophila Ringers on an ice-cold depression slide . The Ringers were then pipetted off, and an ice-cold alcohol solution (98 .5% ETOH, 10 mM acetate buffer, pH 5 .0, 5 mM MgCl2) was added . The paragonia, vas deferens, and other extraneous tissue were dissected away, and the alcoholic solution and testes were transferred to small test tubes and stored at -20°C until enough groups were collected for electrophoresis .
Maden, B . E . H . (1971) . Prog . Biophys. Molec . Biol . 22, 127-177 . Malva, C ., Graziani, F ., Boncinelli, E., Polito, L ., and Ritossa, F . (1972) . Nature New Biol. 239, 135-137 . Melli, M ., and Pemberton, R . E . (1972) . Nature New Biol. 236, 172174 . Meyer, G . F., and Hennig, W . (1974) . Chromosome (Berlin) 46, 121-144 . Miller, L ., and Knowland, J . (1970). J . Mol . Biol . 53, 329-338 . Mohan, J ., and Ritossa, F . M . (1970) . Dev . Biol . 22, 495-512 . Procunier, J . D ., and Williamson, J . H . (1974) . Dev . Biol . 39, 198209 . Puckett, L . D ., and Snyder, L. A . (1974) . Biochem . Genetics 11, 249-260 . Ritossa, F . M . (1968) . Proc . Nat . Acad. Sci . USA 60, 509-516 .
Extraction and Separation of rRNA from Testes rRNA was extracted from Drosophila by a modified method of Tomkins and Billington (1972) . After rinsing the tissue twice in cold E buffer (30 mM NaH 2 PO4 , 1 mM EDTA, 36 mM Tris, pH 7 .5, Loaning, 1969), it was homogenized in a Dual 20, 1 ml capacity all glass homogenizer (Kontes) in 0 .1 ml E buffer made up to 1 % SDS . The homogenate was allowed to stand at 23°C for 2 hr . The homogenate was made up to 6% RNAase-free sucrose (Schwarz/Mann) and centrifuged at 10,000 rpm for 10 min . Then 50 µl of the supernatant was layered over a 2 .5% polyacrylamide gel for electrophoresis . Gels were scanned with a Gilford spectrophotometer at 260 rim, then fractionated in a Gilson automatic gel fractionater. The gel slices were treated with 0 .3 ml 25% H202 and placed at 50°C for 36 hr . Ten ml of Biosolve scintillation fluor (12 g PPO, 200 µg POPOP, 15% Biosolve BBS-3 up to 3 I with toluene) were added to the cooled vials to determine the distribution of radioactivity throughout each gel . Acknowledgments We thank Jean Bertman for excellent technical assistance . We are also grateful to F . Harrison, F. Wilt, and H . Woodland for helpful discussions during the preparation of this manuscript . This research was supported by research grants from NIH and from NSF to B . I . K . This article is from a dissertation submitted by the senior author in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biology .
Ritossa, F ., and Spiegelman, S . (1965) . Proc . Nat . Acad . Sci . USA 53, 737-745 . Ritossa, F., Atwood, K . C ., and Spiegelman, S . (1966a) . Genetics 54, 819-834 . Ritossa, F. M ., Atwood, K . C ., Lindsley, D. L ., Spiegelman, S . (1966b) . Nat Cancer Institute Monogram 23, W . S . Vincent and 0 . L . Miller, Jr ., eds. pp. 449-471 . Tartof, K . D . (1971) . Science 171, 294-297 . Tartof, K . D . (1973) . Genetics 73, 57-71 . Tartof, K . D. (1974) . Cold Spring Harbor Symp . Quant . Biol . 38, 491-500 . Tartof, K . D ., and Perry, R . (1970) . J . Mol. Biol . 51, 171-183 . Tomkins, J . K., and Billington, T . (1972) . Analytical Biochem . 50, 494-499 . Weinmann, R . (1972) . Genetics 72, 267-276 . Williamson, J . H ., Procunier, J . D ., and Church, R. B . (1973) . Nature New Biol . 243, 190-191 .
Regulation in rDNA-Deficient Drosophila melanogaster
A. W . Shermoen* and B . I . Kiefer Department of Biology Wesleyan University Middletown, Connecticut 06457
Summary Bobbed mutants of Drosophila melanogaster were used to determine whether there is any functional compensation for deficiency in rDNA content . The rate of rRNA accumulation was measured in the testes of five bb mutants of different phenotypic severity and in a wild type strain . The different rates of rRNA accumulation were compared to the phenotype (macroscutellar bristle length) and found to have a direct correlation (as in Weinman, 1972) . However, there was not a direct relationship between the rate of rRNA accumulation and rDNA content . It is concluded that there is regulation of rRNA accumulation in some mutants, but that the regulation cannot be considered to be a compensation for the lack of rDNA . These results are discussed relative to other observations on the regulation of RNA synthesis in Drosophila . Introduction Ribosomal RNA has been the subject of many studies on gene activity in eucaryotes because of its accessibility as a primary gene product . Despite the mass of published work on the synthesis and processing of rRNA, we have yet to come to an understanding of the control of its transcription . The analysis of mutants affecting the process of rRNA synthesis is an especially attractive way to investigate the genetic activity of rDNA . Drosophila melanogaster is a choice organism for this approach because of the existence of a large number of mutants which are at least partial deletions of rDNA (that is, bobbed or bb) (Ritossa, Atwood, and Spiegelman, 1966a) . rDNA has been found to be localized to NO regions, which in this species are two in number and reside on the X and on the Y chromosomes (Ritossa and Spiegelman, 1965) . The bb mutants are recognized most easily by their relatively short macroscutellar bristles . They also generally have a thinner cuticle and longer period of development than wild type . While a study of rRNA gene activity may constitute a special case of regulation, there are structural and functional correlations with other genes . Other redundant genes which are known are 5S RNA, *Present address : Department of Zoology, University of California, Berkeley, California 94720 .
tRNA, histone genes, and some genes involved in spermatogenesis in Drosophila (Brown and Weber, 1968 ; Ritossa et al ., 1966b ; Kedes and Birnstiel, 1971 ; and Hennig, 1968) . A further similarity between rDNA and other genes is indicated by evidence that many immediate gene products may be transcribed as a large precursor RNA which is then processed to the smaller product found in the cytoplasm (Melli and Pemberton, 1970 ; Lindberg and Darnell, 1970 ; Maden, 1971 ; Firtel and Lodish, 1973) . A study of rRNA synthesis therefore may provide information concerning both transcriptional and posttranscriptional control mechanisms of gene expression . Two ways in which rRNA synthesis is regulated in Drosophila have already been described . Both mechanisms of regulation involve the increase in the amount of template present for transcription . "rDNA compensation," a single generation increase in rDNA content, is restricted to somatic cells and occurs when rDNA on the X chromosome is opposite another chromosome deficient in rDNA, such as X/O or X/Ybb - males and X/X"o- females (Tartof, 1971, 1973) . [Genetic nomenclature for rDNA mutants in D . melanogaster : bb and bbds = partial deletions ; - bb, bb-, and NO- = virtually complete deletions ; Ybb- has about 30 rRNA genes (Tartof, 1973) .] "Magnification", on the other hand, occurs in germ cells . When a bb chromosome is maintained over several generations opposite another bb chromosome, such as Xbb / Ybb there is a gradual increase of rDNA so that Xbb reverts to Xbb+ (Ritossa, 1968 ; Tartof, 1974) . Previous work measuring the rate of rRNA synthesis in ovaries relative to the rDNA content of the fly was interpreted to show an unmediated use of the rDNA in bb mutants (Mohan and Ritossa, 1970 ; Weinman, 1972) . These authors concluded that there was a constant rate of rRNA synthesis/gene for all flies which contained up to the haploid amount of rDNA . Flies containing a greater than haploid rDNA content did not have an increase in rRNA synthesis (Mohan and Ritossa, 1970 ; Krider and Plaut, 1972) . These investigations would all seem to indicate that in Drosophila, a primary method of regulating rRNA production lies at the level of altering the template content of the cell . There are, of course, other possibilities of control . The work reported here involved the use of bobbed flies whose phenotype varied over a wide spectrum . Two questions were of primary concern : -Is there regulation of rRNA synthesis in rDNA-deficient mutants? -If such regulation exists, is it correlated with the amount of rDNA present?
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Our experiments show that a model of rRNA regulation in Drosophila melanogaster based solely upon the amount of ribosomal genetic material present is too simplified .
by others (Tartof, 1971 ; Williamson, Procunier, and Church, 1973) and was roughly four times the rDNA content of the bb genotypes, which clustered around 100 genes . ybb Su Var 5 and Y bb- chromosomes have previously been measured and shown to be equivalent in rDNA content (Malva et al ., 1972) . The first question asked was whether the bristle length is directly representative of the rDNA content. As Figure 1 shows, the answer is no . It is apparent that the bristle length-rDNA content relationship does not hold for wild type relative to bb, and it is also evident that no such relationship exists among the bb mutants . This is the first indication that there may be a regulation of rDNA cistrons in bb flies . Next the rates of rRNA accumulation were measured for the testes of each of the genotypes, and are represented as specific activities . Use of the mass (µg) of rRNA as standardization for the dpm accumulation by 18S and 28S is legitimate since measurement of the RNA/testis revealed that the most severe bb (car bb/Ybb - ) contained only 20% less RNA/organ than did wild type (0 .84 µg/testis and 1 .05 µg/testis, respectively) . The differences in these bb specific activities therefore would be somewhat higher than the real rate of accumulation, but the difference is insignificant relative to the degree of change which has been measured . Another way in which the specific activities are verified as being truly representative of the rates of rRNA accumulation (and probably synthesis) is that the amount of accumulation of radioactivity as tRNA (as
Results Measurements of the posterior macroscutellar bristles are shown in order of their increasing length in Table 1 . These genotypes were selected because their bb phenotype varied in severity . One could therefore determine not only whether there is regulation of rRNA synthesis, but also whether that regulation is related to the rDNA content . The rDNA content of each genotype was measured by saturation hybridization of 3 H-rRNA to DNA isolated from adult males (Table 2) . The wildtype rDNA content is approximately that reported Table 1 . Macroscutellar Bristle Lengths
(µ)
Genotype
Bristle Length
y w bbd•/ Ybb -
282
Standard Deviation 33
car bb/Ybb-
285
16
gt bb'T/Ybb -
326
8
y w bbd•/ Y - bb
345
24
y w bbd•/ Ybb Su Var 5
391
27
Samarkand+ iso
365
19
The posterior dorsal thoracic (macroscutellar) bristles were measured by an ocular micrometer in a dissecting microscope . The males measured were from the cultures used to obtain DNA for the hybridization experiments .
Table 2 . 3H-rRNA-DNA Saturation Hybridization Levels Genotype
rRNA hybridization x 100 DNA
Standard Deviation
Gene Number
y w bbd•/ Ybb -
0 .0889
0 .0026
101
car bb/Ybb-
0 .0783
0 .0118
89
gt bbl /Ybb-
0 .0805
0 .0041
92
y w bbd•/ Y - bb
0 .1076
0 .0171
123
y w bbd•/ Ybb Su Var 5
0 .0915
0 .0166
104
Samarkand+ iso
0 .3555
0 .0387
405
Determination of rDNA content was made by rRNA-DNA hybridization (Gillespie and Gillespie, 1971) . A minimum of six determinations were made for each saturation point . Blank filters and filters containing E . coli DNA were used to monitor background radioactivity . The calculation of rRNA gene number was made in the following way : rRNA specific activity 75,700 cpm/µg
DNA/filter 5 .96 µg
rRNA cpm filter 414 cpm
414 cpm = 0 .00547 µg rRNA 75,700 cpm/µg 0 .00547 µg rRNA x 100 = 0 .0918% hybridization 5 .96 µg DNA 0 .0918% x 2 = 0 .1836% rDNA (2 .4 x 10 1 1 daltons EDNA) (1 .836 x 10-3 rDNA) _ 104 .8 genes 4 .2 X 106 daltons rDNA
diploid DNA 2 .4 x 10 11 daltons
18S + 28S 2 .1 x 106 daltons
Regulation in rDNA-Deficient D . melanogaster 277
seen by 10% polyacrylamide gel electrophoresis) is similar for genotypes which vary widely in rDNA (car bb /Y bb- , Sam++ iso) (Mohan and Ritossa, 1970 ; Clark, 1975) . These results indicate both that the precursor pools have very similar specific activities and that the same amount of DNA is represented in all of the tissues tested . Furthermore, possible differences in time of pool equilibration were compensated for by subtracting the dpm accumulated in the first hour of incorporation from the dpm incorporated after 3 hr . Figure 2 is a plot of the rate of radioactive rRNA accumulation compared with the bristle length and shows a direct relationship between the two parameters . This is the same result as Weinmann (1972), who used different conditions including an isogenic background for testing single X chromosomes . However, the substantive relationship (for the question of rate regulation) is shown in the plot of the rate of rRNA synthesis relative to the rDNA content (Figure 3) . The rate of rRNA synthesis is different for wild type than for the bb flies, and even within the series of bb mutants there is not a clear direct relationship. The rate problem is put in clearer perspective if one divides the rate of accumulation by the gene number and relates the
rate of accumulation/gene to the rDNA content . If there is a direct relationship of the rate of rRNA accumulation to rRNA gene number, this graph will have a straight line parallel to the abscissa . This is clearly not the case (Figure 4) . Discussion In agreement with Weinmann (1972), the data shown here (Figure 2) demonstrate a direct relationship between the rate of radioactive rRNA accumulation and bristle length . This agreement in results comes despite the care taken by Weinmann to use a completely isogenic background changing only the tested chromosome . The similarity is striking for two reasons . First, Weinmann used two chromosomes that were also used in this study, gt bbl 1 and car bb ; if the ratio of relative rates for each genotype ( 9a b,,°,) of Weinmann's data (Table 3, Weinmann, 1972) is compared with the data here they correspond well within experimental error, 0 .49 and 0 .52, respectively . In addition, the correlation shown here comes from the rate of accumulation as measured in spermatogonia and primary spermatocytes, whereas Weinmann's data uses the rate
500
y w bbd,
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L Q)
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O X E
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Ybb
Ybb 100
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ST i
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car bb Y bb -
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Ybb
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Su Var 5 i
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I
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I
I
I
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A
I
280
L
I
320
i
L
360
1
1
400
Bristle length ()u)
420
Bristle length (,u) Figure 1 . Bristle Length Relative to rRNA Gene Number The macroscutellar bristle lengths (Table 1) are graphed relative to the rRNA gene number (Table 2) for their respective genotype . The bb genotypes do not have a linear relationship .
Figure 2 . A Plot of Bristle Length Relative to rRNA Accumulation The relationship of macroscutellar bristle length is shown relative to the rate of radioactive rRNA accumulation in the testes for each genotype (see Experimental Procedures for details) . Isotope incorporation is standardized to the rRNA content of each genotype (see Results section for a discussion of this use of specific activity) .
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of accumulation in whole female D melanogaster . Both of these sets of data are related to the product of a third cell type, the trichogen cells that produce the bristles . These tissue differences imply that if regulation of rRNA synthesis is found it will be due to something other than a cytoplasmic feedback mechanism, since one would expect to find a variety of physiological conditions in these disparate cell types. Figures 3 and 4 show there is regulation of rRNA accumulation, and that this regulation of the efficiency of expression of the rRNA genes is not a function of the number of rRNA genes present . These data contradict the interpretation of their data by Mohan and Ritossa (1970), who maintained that there is a constant rate of rRNA synthesis through the "haploid" rDNA content . Miller and Knowland (1970) found a somewhat lower than wild type rate of rRNA synthesis/gene in a partial deletion of rDNA in Xenopus laevis, again indicating a correlation between the number of rRNA genes and accumulation of rRNA when rDNA is below some threshold level . Although our data indicate that some form of regulation is occurring, it is not yet possible to clearly distinguish between transcriptional and posttranscriptional control in these studies . Some restrictive regulation of rRNA synthesis in Drosophila has previously been discovered in addition to work by
Mohan and Ritossa (1970) . Krider and Plaut (1972) found that the rate of incorporation of 3 H-U by salivary gland nucleoli is identical for genotypes of one to four intact NOR and that, seemingly, no more than two NOs are active at any one time . Second, Meyer and Hennig (1974) counted active rRNA cistrons from EM spreads and concluded that only about half of the NOR are active in primary spermatocytes of Drosophila hydei . They also think that it is likely that not all of those cistrons which eventually function are active in early primary spermatocytes . Finally, Baker (1971) and Puckett and Snyder (1974) showed that there are some sct inversions (that is, an inversion which includes one break point adjacent and proximal to the NOR) with a full complement of rDNA which is subject to position-effect restriction . [Position-effect variegation is considered to be a restriction of the transcriptive activity which occurs in some cases of translocations and inversion of euchromatin to heterochromatin (Baker, 1968) .] Two interesting observations relative to positioneffect variegation (PEV) and its possible relationship to the rDNA region may be mentioned here . Many studies have demonstrated that PEV can be suppressed by the addition of heterochromatin . Brosseau (1964) demonstrated that there were two regions of the heterochromatic Y chromosome which were particularly effective in overcoming PEV 500
y w bbd,
50
. 1
i ,~i y w bbds
Y bb Su Var 5
Ybb Su Var 5 450
Sam' ISO 45
F-~1
40
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y w bbds y -bb
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y w bbds y -bb
a, C
fr1
O
p
X
4H gt bb" y b b-
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gt bb"
0+ Iw
?
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y bb -
25
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E C1. 0
E . 0 -0 20
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car bb ybb -
car bb "JV Ybb-
y w bb Y bb
d'
y w bbds ybb I+-1
y Sam + ISO
100
10 50 5
0 .1 0 .1
0.2 rRNA / DNA x
0.3
0 .2 rRNA /DNA x
0.3
0 .4
100
0 .4
100
Figure 3 . A Plot of rRNA Gene Number to rRNA Accumulation The saturation level of rRNA hybridization is graphed against 3H rRNA accumulation .
Figure 4 . A Plot of rRNA Gene Number to rRNA Accumulation/ Gene This graph demonstrates the average rate of utilization of rRNA genes for each genotype . Two bb mutants and wild type have the same average rates of utilization .
Regulation in rDNA-Deficient D . melanogaster 2 79
of an X chromosome locus . These two regions were located near the fertility loci, kl-1 and ks-1 . ks-1 is adjacent and distal to the NOR on the Y chromosome . Second, it is known that rearrangements of the It locus, located in heterochromatin of chromosome 2, also express a PEV . However, this heterochromatic rearrangement [R(/t+)] responded oppositely to added Y chromosomes as compared to a euchromatic rearrangement [R(w+)] . That is, It+ was most active (normal) in the X/O condition, and progressively decreased its activity with addition of heterochromatin (Baker and Rein, 1962) . Hence, those heterochromatic regions which are so successful in restoring gene activity to R(euchromatin) seem to restrict the activity of at least one example of R(heterochromatin) . These various examples of transcriptive regulation in Drosophila melanogaster suggest several conclusions in light of the data presented in this report . (a) Because there are examples of transcriptive regulation and a lack of natural examples for the regulation of RNA processing, it is simplest to interpret the increased rate of rRNA accumulation for three genotypes, y w bbds/Y - bb, gt bbl l/Ybb-, and y w bbds/Ybb Su Var 5 as an increase in the average rate of rRNA synthesis/gene . Since the change in the rate of rRNA synthesis is not correlated with the rDNA content of the fly, the explanation proposed to account for the observed rate differences is that there is a region external (and adjacent) to the rDNA which has affected the utilization of the rRNA gene . This suggestion also has been made by Procunier and Williamson (1974) from work on temperature sensitive bb mutants . Furthermore, the normal (wild type) condition of rDNA activity is under restriction, and it is only in some mutants in which this restricted transcription has been alleviated . (b) Second, those chromosomes which display more efficient use of their rDNA may have rearrangements within the heterochromatin which either separate the rDNA from those regions which suppress euchromatin PEV or destroy, at least in part, such regions . One prediction from the first proposal would be that since an X chromosome bb mutant did show an increase in rRNA synthesis/gene (gt bb"), and Baker (1971) found some sct chromosomes in which rRNA synthesis is depressed (thereby eliminating the proximal side of the NOR from consideration), we would postulate that some sc 4 inversions (breakpoints distal to the NOR) would show an increase in rRNA synthesis/gene . A prediction from the second proposal would be that Ybb chromosomes which have an increased
rate of rRNA synthesis/gene should have the same or decreased ability to suppress PEV . These and more direct experiments relative to the nature of the regulatory mechanism(s) involved are currently being tested . Experimental Procedures Drosophila Stocks A list of the crosses and the progeny used in these experiments is provided below . All genotypes were obtained from I . I . Oster at Bowling Green University . Cross
F, Used
wm4 /Ybb- x ywbbds/ywbbd ,
ywbbds/Ybb -
wm4/Ybb- x car bb/car bb
car bb/Ybb -
wm•/ Ybb - X gt bb""/c1 B
gt bbl I / Ybb-
w sn bb/Y-bb x y w bbd'/y w bbd ,
y w bbd,/Y-bb
wm4/Ybb Su Var 5 x y w bbd,/y w bbds
y w bbds/Ybb SU Var 5
Samarkand isogenic Wild type
Sam+ iso
DNA Extraction DNA was extracted by modification of a method developed by L . Weber (personal communication) . At least 5 g of adult males of any one genotype were ground to a fine powder with a dry icecooled mortar and pestle . The powdered flies were slowly stirred into 9 vol of room temperature NETM buffer (0 .1 M NaCl, 0 .01 M EDTA, 0 .03 M Tris, 0 .01 M 8-mercaptoethanol, pH 8 .0) with Triton X-100 added to 0 .5% . The mixture was then homogenized twice with a Dounce glass homogenizer and "B" pestle . Cuticle and large tissue masses were sedimented with a hand centrifuge, and then the supernatant was centrifuged at 5000 rpm for 10 min in the SS-34 Sorvall rotor . The nuclear pellet was resuspended in 5 vol of NETM buffer and homogenized as before . The nuclei were sedimented again and resuspended in T E buffer (0 .1 M Tris, 0 .1 M EDTA, pH 8 .4). SDS was added to a concentration of 2%, and then the solution was shaken gently for 20 min . 5 M NaCl 04 was added to a final concentration of 1 M NaCl 04 . The mixture was shaken gently for 20 min . The DNA was then extracted once with 1 vol buffer saturated neutralized phenol, and twice with 1 vol of chloroform/isoamyl alcohol (24 :1, v/v) . DNA was precipitated with 2 vol 95% ethanol and resuspended in 1 x SSC. The DNA solution was treated with 100 µg/ml pancreatic ribonuclease X11-A (Sigma), 20 units/ ml T1 ribonuclease (Worthington), and 200 µg/ml a-amylase (Sigma) for 2 hr . Pronase B (Calbiochem), preincubated for 4 hr at 37°C, was added to 50 pg/ml and incubated for an additional hour . DNA was then extracted 3 times or more with equal volumes of chloroform/isoamyl alcohol until the 260/280 ratio was 1 .95 or greater. The DNA was precipitated with 2 vol of 95% ethanol . DNA was resuspended in 0 .1 x SSC and centrifuged at 42,000 rpm in an SW-50 .1 Spinco rotor for 30 min in order to sediment any remaining polysaccharides. All DNA solutions had a 260/230 ? 2 . Labeling and Extraction of rRNA for Hybridization (Modification from Ritossa et al ., 1966a.) Samarkand strain embryos were collected from a yeast plate that had been in a population cage for 3-4 hr . Two-tenths g of embryos were placed on 21 ml of medium in a crystallizing dish (10 cm diameter x 5 cm height) . The medium contained 16 ml of mixture A [100 ml distilled H 2O, 10 g Instant Drosophila Medium (Carolina Biological), 2 g Fleischmans Yeast, 0 .8 g Bacto agar] and 5 ml uridine-5- ; H (1 me/ml ; 28 curies/mM ; New England Nuclear) . The dishes were covered with cheesecloth and kept at high humidity and 25°C . After 1 week the larvae were collected on nylon stocking (Kirsten) and washed with distilled water . The rRNA was extracted according
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to Tartof and Perry (1970), and the 28S and 18S rRNA collected individually from 5-20% sucrose gradients . Hybridization was done using 28S and 18S in 2 :1 ratio by UV absorbance . The rRNA specific activity was 75,700 cpm/µg . Hybridization Procedures DNA was denatured by alkali or by boiling for 10 min in 0 .1 x SSC, then adsorbed to 25 mm nitrocellulose membrane filters (Schleicher and Schuell, type B-6) according to the methods of Gillespie and Spiegelman (1965) . The amount of DNA adsorbed to the filters was determined by measuring the A260 prior to filtration, and then the A 260 of the filtrate, and by the retained radioactivity of radioactively labeled E . coli DNA and S . purpuratus DNA (gift of Dr . C. Alfageme) . In most cases "minifilters" (5 mm diameter) punched from the large filters were used in hybridization . Filters containing radioactive DNA were tested for an even distribution of DNA over the surface of the filter . Minifilters contained from 5-10 µg/filter . The hybridization and posthybridization process methods used were esentially those of Gillespie and Gillespie (1971), with the modification that the incubation medium was 50% formamide (Eastman Kodak Co.)-2 x SSC, (pH 7 .0), and 0 .2-0 .5% SDS . Radioactivity retained as hybrid was counted in a N/C liquid scintillation spectrophotometer for a time that resulted in no more than a 1 % counting error .
Received November 11, 1974 ; revised December 23, 1974 References Baker, W . K . (1968) . Advan . Genet. 14, 133-169 . Baker, W . K . (1971) . Proc . Nat. Acad . Sci . USA 68, 2472-2476 . Baker, W . K ., and Rein, A. (1962) . Genetics 47, 1399-1407. Brosseau, G . E. (1964) . Genetics 50, 237 . Brown, D . D ., and Weber, C . S . (1968) . J . Mol. Biol . 34, 661-680 . Carlson, E . A., and Oster, I . I . (1962). Genetics 47, 561-576 . Clark, S . H . (1975) . Ph .D . Thesis, Wesleyan University, Middletown, Connecticut . Firtel, R . A ., and Lodish, H . F . (1973) . J . Mol . Biol . 79, 295-314 . Gillespie, S ., and Gillespie, D . (1971) . Biochem . J . 125, 481-487 . Gillespie, D ., and Spiegelman, S . (1965) . J . Mol . Biol. 12, 829-842 . Hennig, W . (1968) . J . Mol . Biol . 38, 227-239 . Kedes, L. H ., and Birnstiel, M . L . (1971) . Nature New Biol . 230, 165-169 . Krider, H ., and Plaut, W . (1972) . J . Cell Sci . 11, 675-687 . Lindberg, U ., and Darnell, J . (1970) . Proc. Nat . Acad. Sci . USA 65, 1089-1096. Loening, U . E . (1969) . Biochem . J . 113, 131-138 .
Measurement of rRNA Synthesis Incorporation of radioactive isotope : 2-3 H-adenosine (17 .7 curies/ mM, New England Nuclear) was lyopholized and resuspended in sterile Drosophila Ringers at a concentration of 20 me/ml . Approximately 0 .25 µl (5 µc) was injected into the abdomen of each lightly etherized fly with a hand-pulled glass needle (Carlson and Oster, 1962) . After 1 or 3 hr of incorporation, the flies (30 flies/group) were frozen in a vial on dry ice . The testes were dissected into ice-cold Drosophila Ringers on an ice-cold depression slide . The Ringers were then pipetted off, and an ice-cold alcohol solution (98 .5% ETOH, 10 mM acetate buffer, pH 5 .0, 5 mM MgCl2) was added . The paragonia, vas deferens, and other extraneous tissue were dissected away, and the alcoholic solution and testes were transferred to small test tubes and stored at -20°C until enough groups were collected for electrophoresis .
Maden, B . E . H . (1971) . Prog . Biophys. Molec . Biol . 22, 127-177 . Malva, C ., Graziani, F ., Boncinelli, E., Polito, L ., and Ritossa, F . (1972) . Nature New Biol. 239, 135-137 . Melli, M ., and Pemberton, R . E . (1972) . Nature New Biol. 236, 172174 . Meyer, G . F., and Hennig, W . (1974) . Chromosome (Berlin) 46, 121-144 . Miller, L ., and Knowland, J . (1970). J . Mol . Biol . 53, 329-338 . Mohan, J ., and Ritossa, F . M . (1970) . Dev . Biol . 22, 495-512 . Procunier, J . D ., and Williamson, J . H . (1974) . Dev . Biol . 39, 198209 . Puckett, L . D ., and Snyder, L. A . (1974) . Biochem . Genetics 11, 249-260 . Ritossa, F . M . (1968) . Proc . Nat . Acad. Sci . USA 60, 509-516 .
Extraction and Separation of rRNA from Testes rRNA was extracted from Drosophila by a modified method of Tomkins and Billington (1972) . After rinsing the tissue twice in cold E buffer (30 mM NaH 2 PO4 , 1 mM EDTA, 36 mM Tris, pH 7 .5, Loaning, 1969), it was homogenized in a Dual 20, 1 ml capacity all glass homogenizer (Kontes) in 0 .1 ml E buffer made up to 1 % SDS . The homogenate was allowed to stand at 23°C for 2 hr . The homogenate was made up to 6% RNAase-free sucrose (Schwarz/Mann) and centrifuged at 10,000 rpm for 10 min . Then 50 µl of the supernatant was layered over a 2 .5% polyacrylamide gel for electrophoresis . Gels were scanned with a Gilford spectrophotometer at 260 rim, then fractionated in a Gilson automatic gel fractionater. The gel slices were treated with 0 .3 ml 25% H202 and placed at 50°C for 36 hr . Ten ml of Biosolve scintillation fluor (12 g PPO, 200 µg POPOP, 15% Biosolve BBS-3 up to 3 I with toluene) were added to the cooled vials to determine the distribution of radioactivity throughout each gel . Acknowledgments We thank Jean Bertman for excellent technical assistance . We are also grateful to F . Harrison, F. Wilt, and H . Woodland for helpful discussions during the preparation of this manuscript . This research was supported by research grants from NIH and from NSF to B . I . K . This article is from a dissertation submitted by the senior author in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biology .
Ritossa, F ., and Spiegelman, S . (1965) . Proc . Nat . Acad . Sci . USA 53, 737-745 . Ritossa, F., Atwood, K . C ., and Spiegelman, S . (1966a) . Genetics 54, 819-834 . Ritossa, F. M ., Atwood, K . C ., Lindsley, D. L ., Spiegelman, S . (1966b) . Nat Cancer Institute Monogram 23, W . S . Vincent and 0 . L . Miller, Jr ., eds. pp. 449-471 . Tartof, K . D . (1971) . Science 171, 294-297 . Tartof, K . D . (1973) . Genetics 73, 57-71 . Tartof, K . D. (1974) . Cold Spring Harbor Symp . Quant . Biol . 38, 491-500 . Tartof, K . D ., and Perry, R . (1970) . J . Mol. Biol . 51, 171-183 . Tomkins, J . K., and Billington, T . (1972) . Analytical Biochem . 50, 494-499 . Weinmann, R . (1972) . Genetics 72, 267-276 . Williamson, J . H ., Procunier, J . D ., and Church, R. B . (1973) . Nature New Biol . 243, 190-191 .