Cell, Vol. 15,1087-1093,

November

1978. Copyright

Q 1978 by MIT

Genetic and Molecular Organization of the 5s Locus and Mutants in D. melanogaster James D. Procunier Department of Zoology The University of British Columbia Vancouver, Canada Robert J. Dunn Department of Biochemistry The University of British Columbia Vancouver, Canada Summary The topography of an entire redundant locus was analyzed by both genetic and molecular means. Three mutants (mine, min’, minz) allellc to the 5s rRNA genetic locus on chromosome 2 of D. melanogaster were isolated. Flies exhibit a mutant phenotype when hemlzygous for a min allele, but flies having two doses are wild-type. Saturation hybridization experiments show that the alleles are gross deficiencies each deleting an equal amount of 5s DNA. Each of the three mutant min alleles produces a dlstlnct temperature-sensitive viability phene, and thus they are suggested to be pseudoalleles within the same redundant locus. Using the segmental aneuplold method (Lindsley et al., 1972), the 5s gene cluster was subdivided into proximal and distal halves. Both saturatlon hybridization experiments and genetic tests show that each half contains about eighty 5s genes. The complementation of the min alleles with the proximal and distal halves of the cluster indicates that both halves function independently. We present evidence which supports the model that all of the 160 5s genes are arranged as a single continuous cluster of tandem repeats with no large interdispersive DNA segments not complementary to 5s rRNA. Introduction The 5S rRNA locus of Drosophila melanogaster provides an ideal model system for studying the structure, function and control of a eucaryotic redundant locus. The localization of these genes to a single euchromatic site easily facilitates the isolation, recombination, cytological examination and molecular analysis of 5s rRNA mutants. The 160 copies of the 5s rRNA gene per haploid genome are localized at 56F on chromosome 2R by both in situ hybridization (Wimber and Steffensen, 1970; Grigliatti et al., 1973) and genetic evidence (Procunier and Tartof, 1975). The molecular organization of the Drosophila 5S genes has been reported (Procunier and Tat-tof, 1976; Artavanis-Tsakonas et al., 1977; Hershey et al., 1977). These genes occur in a tandemly repetitious arrangement with the

repeating unit comprising a G+C-rich structural gene and an A+T-rich spacer sequence. The spacer sequences display heterogeneity in length and are presumably not transcribed. Although most reports concerning the physical organization of redundant loci deal only with a few gene repeats, the organization of an entire cluster is unknown. This report gives a genetic and molecular description of all the 160 5S genes of the fruit fly. Contrary to a previous report (Procunier and Tartof, 1976), the entire 5S gene cluster is bounded by Eco Rl restriction endonuclease sites and has no internal site. This result is supported by Artavanis-Tsakonas et al. (1977), who recently suggested a single continuous cluster of 5S genes for Drosophila with no internal Eco Rl , Bg 1, Bg 11 or Barn restriction sites. Three 5S rRNA mutants, which are gross deletions of 5S DNA, reduce the length of the wild-type Eco Rl fragment by an amount proportional to the size of the deletion. This result is in agreement with the single cluster model. Results Genetic Analysis of the 5s rRNA Locus Using the segmental aneuploid method (Lindsley et al., 1972), the 56F region can be subdivided into a series of smaller segments, each of which can be genetically duplicated or deleted (Figures 1 and 2). The T(Y;2)L139 breakpoint lies to the left of the 56F region (56E), T(Y;2)L62 within the 56F region and T(Y;2)L141 to the right of the 5S site (56F). Previous studies showed that the majority, if not all, of the 5S genes are localized between the L139 and L141 breakpoints (Procunier and Tartof, 1975). One dose of the 56F region has enough 5S genes for a wildtype phenotype since +/Df L139-L141 flies are indistinguishable from Oregon-R. In addition, the Df L139-L141 chromosome does not uncover any of the known Minute loci near this region (Lindsley et al., 1972). The eucaryotic ribosome contains equimolar amounts of 5,18 and 28s RNA. Since bobbed (bb) mutants (short, thin bristles, abdominal etching and semi-lethality) were shown to be partial deletions of 18 and 28s rRNA genes on either the X or Y chromosome nucleolar region (Ritossa, Atwood and Spiegelman, 1966; Procunier and Williamson, 1974), 5S rRNA mutants of a similar phenotype were screened for at the 56F region. Two triethylenemelamine-induced mutants (miff’ andmin’) and one spontaneous mutant (min”) were recovered by showing allelism to the Df L139-L141 chromosome. Flies of the genotype min/Df L139-L141 have a min phenotype, whereas flies homozygous for each allele (minlmin) are wild-type, demonstrating the additivity of two alleles. Flies heterozygous for any

Cell 1088

Phyricol

map

Genetic

map

Dp or DfLl39-Ll41 Dp or DfLl39-L62

Dp or DfL62-Ll41

I

I I

1 I

Figure

1. Physical

J

and Genetic

The solid bars represent the represent the duplications or position of the translocation sites are indicated. The three one of three possible deletions.

Map of the 5s rRNA Gene

Cluster

160 55 gene copies; the open bars deficiencies. The designation and breakpoints and Eco Rl restriction min alleles are positioned as any

combination of the three alleles are wild-type. Genetic evidence indicates that the translocation L62 breakpoint splits the 5s gene cluster into two approximately equal segments (Figure 1). Since the breakpoint lies between the L139 and L141 breakpoints, the combined length of the proximal deficiency (Df L139-L62) and the distal one (Df L62L141) is the large deficiency (Df L139-Ll41). Flies of the genotypes minlDf Ll39-L62 and minlDf L62L141 are wild-type for all the three min alleles. Thus both deficiencies Ll39-L62 and L62-L141 have a sufficient number of 5s genes to revert the min phenotype to wild-type. Since these contiguous deficiencies together span the entire 5s rRNA gene cluster, their common breakpoint (L62) must split the 5s gene cluster. The complementation of the min alleles with either deficiency demonstrates that the 5s gene cluster can be split near its midpoint and that each segment is independently functional. Figure 3 shows the viability of flies heterozygous for various min alleles and the Df L139-L141 chromosome at different temperatures. All three alleles reduce viability at 29”C, flies bearing the min’ and min2 alleles being the least viable with respect to the wild-type allele. The min’ and min2 alleles, however, also cause a greater reduction in viability at all temperatures. The three mutant alleles are heat-sensitive but show different viability responses. The minlDf Ll39-L141 flies demonstrate a reduced rate of development, eclosing 2-3 days after min+/Df L139-L141 flies. The bristle phenotype of flies homozygous and heterozygous for the three alleles was normal at all temperatures. To demonstrate that the different viability responses

to temperature of the three alleles are due to mutations in the 5s rRNA cluster per se, possible second site mutations on the min chromosome must be ruled out. The viability of flies heterozygous for the min chromosomes and Df L139-L62 chromosomes was wild-type (unpublished data), thus eliminating a second site recessive, heat-sensitive semi-lethal proximal to the 5s rRNA cluster and allelic to Df Ll39-L141. Similarly, viability of minlDf L62-L141 flies was normal, thereby eliminating the possible second site mutant distal to the 5s rRNA genes. Furthermore, the reduced ratio of minlDf L139-L141 males to their minlCy sisters observed in Figure 3 rules out a second site dominant, temperature-sensitive, semi-lethal on themin chromosomes. Thus these three mutants show different temperature-sensitive viability phenes and are the result of unique mutations in the 5s rRNA cluster.

5s rRNA Gene Numbers The 5s rRNA-DNA hybridization technique used in these experiments, where all components are in solution, gave very similar results to the classical membrane filter method (Procunier and Tartof, 1975; Weber and Berger, 1976). This result enhances the creditability of measuring actual 5s gene number values by hybridization. Wild-type DNA from D. melanogaster males and females, which has two doses of the 56F region, has approximately 162 5s genes per 5s region (Table 1 and Figure 2). Males and females have an equal number of 5s genes (Tartof and Perry, 1970). The genotype +/Dp Ll39-L141 has three doses of the 5s genes and 496 5s genes, each dose of 5s genes contributing about 166 genes. The 5s gene multiplicity is directly proportional to the number of 56F regions; this ensures that the majority of the 5s genes reside at this site. Molecular hybridization results confirm the genetic evidence that the 5s gene cluster is split into two equal segments by the translocation L62 breakpoint. Both +/Dp Ll39-L62 and +/Dp L62L141 genotypes have an equal number of 5s genes. Thus both the Dp L139-L62 and Dp L62-L141 segments have eighty 5s genes (Table 1). Since the corresponding deficiencies, Df Ll39-L62 and Df L62-L141, are reciprocal combinations of the duplications of the initial stocks, it can be deduced that both are deleted for about eighty 5s genes. The Df Ll39-L62 extends from the midpoint of the 5s gene cluster to the proximal end to delete eighty 5s genes and proximal non-5S DNA. Similarly, Df L62L141 deletes the distal eighty 5s genes and extends through the distal boundary of the 5s gene cluster. Table 2 shows the number of 5s genes for the various min alleles, measured as minlmin males and females. The min alleles represent a 56-65%

5s rRNA 1089

Genes

3

Figure 2. Diagrammatic ious Doses of 55 Genes

Representation

of Genotypes

with

Var-

The open bars represent the 5s gene cluster and the straight lines are adjacent DNA not coding for 5s rRNA of chromosome 2. The wavy lines indicate centric heterochromatin of the translocated Y chromosome.

deletion of a 5s gene cluster, and when present in a hemizygous condition (min/Df L139-L141), the adult fly is mutant. The complementation of the various min alleles with either the Df L139-L62 or Df L62-L141 chromosomes can be explained as the presence of a sufficient number of 5S genes, contributed by the deficiency and min chromosomes, for a wild-type phenotype. This explanation applies to the wild-type phenotype of flies homozygous or heterozygous for any combination of the three min alleles.

Restriction

Map of the 5S rRNA Locus

Wild-type D. melanogaster DNA was cut with the restriction endonuclease Eco Rl, and the resulting fragments were separated on 0.4% agarose gels. The fragments were denatured, transferred to nitrocellulose filters and hybridized with 1251-labeled 5s rRNA. Scintillation autoradiography showed a band >49 kb (Figure 4). The molecular size of this band is difficult to estimate accurately due to the fact that the calibration curve plot of log (molecular weight) against mobility is highly nonlinear above 49 kb. Fragments >49 kb migrate to about the same position in this electrophoresis system. Artavanis-Tsakonas et al. (1977) suggested that the Eco Rl 5s DNA fragment from wild-type DNA is about 99 kb; the same difficulty in sizing fragments in this range, however, exists for their electrophoresis system. The previous estimate of 21 kb (Procunier and Tartof, 1976) was inaccurate for the same reasons. The repeat unit size (gene plus spacer) estimated from two independent sources is370220 bp (Artavanis-Tsakonas et al ., 1977; Hershey et al., 1977). On the basis of saturation hybridization experiments, there are 160 copies of the repeat unit per 56F site, thus requiring 59 kb of 5s DNA. The length of the wild-type 5s DNA segment bounded by Eco Rl sites observed in these experi-

ments is within the range of the calculated 59 kb fragments. The Eco Rl 5s fragments from the three min alleles are approximately the same size, corroborating the hybridization data that these alleles have the same number of genes. These deficiencies delete about 58-65% of the entire 5s gene cluster and yield Eco Rl fragments of 21 kb. This is enough 5s DNA to code for about 55 genes given 370 bp for each repeat. An independent measurement of the total number of 5s genes permin allele was carried out by saturation hybridization experiments which also gave 57-69 copies. The close agreement of these two estimates gives creditability to the two independent techniques of determining gene numbers. In addition, it suggests that the 5s DNA of the min chromosomes has no large segment of DNA which is not complementary to 5s rRNA.

Discussion A preliminary attempt to dissect genetically the 5s rRNA locus involved the isolation of mutants in this region. Three 5s rRNA mutants (mine, min’ and min’) were isolated and shown by saturation hybridization experiments to be deficiencies of 5s DNA of approximately equal size. These mutants delete about 58-65% of the locus and when present as one dose (min/Df L139-L141), produce a viable fly with a mutant phenotype. Similarly, deletions of about one half the 16 and 28s rRNA genes on either the X or Y chromosome nucleolar region (NO) yield viable bb flies when present in a hemizygous condition. The three min mutant alleles show quite distinct temperature-sensitive viability phenes which, as shown by genetic tests, are the result of deleted 5s DNA per se, not of other possible second site mutants. The simplest explanation for these allelic differences is that the deficiencies delete different regions of the 5s DNA cluster. There are various examples demonstrating that individual genes within the same genetic cluster differ with respect to transcriptional activity. Scheer, Trendelenburg and Franke (1976) found fully and partially transcribed genes in Xenopus followed by totally untranscribed genes on the same nucleolar axis of the amplified 18 and 28s rDNA. Miller and Knowland (1972) demonstrated that the nucleolar genes of partial deficiency mutants are not transcribed with the same efficiency as the genes of an intact nucleolar organizer in Xenopus. They suggested the presence of DNA sequences in the NO which control the transcription of a large block of rRNA genes so that a deletion of a site could eliminate or reduce the activity of a large number of genes. Shermoen and Kiefer (1975) showed that the rate of 18 and 28s rRNA accumulation for various bb

Cell 1090

Table

1. Number

Genotype of Chromosome 2 +I+

Temperature (“C ) Figure The

3. Temperature

Sensitivity

of min Alleles

of males, X; min/Df L139-L141, to their sisters, s/Y; min/Cy, was taken as an index of relative viability at different temperatures. The standard deviation for each point is indicated. The Df L134L141 chromosome is deleted for all 5s genes, whereas the Cy chromosome has one dose of 5s genes. (C--O)

min’;

ratio

(C-W)

min’;

(0-O)

min?

(-0)

min+.

mutants of D. melanogaster is not a function of the number of rRNA genes present. The existence of different 5s structural gene sequences (thus heterogeneous RNA species) does not explain the different min phenotypes at least for the miff2 allele, since the 5s rRNA sequences from wild-type and minZ DNA are identical (B. Jordan, personal communication). Although we prefer to explain the phenotypic differences of the min alleles by differential transcriptional activity of various 5s genes, other possibilities might exist. For example, the total number of 5s genes of the various alleles might be slightly different, but not detected by our hybridization experiments, and might result in distinct temperature-sensitive viability phenes. The fact that the T(Y;2)L62 breakpoint splits the 5s gene cluster near its midpoint provides a means of genetically synthesizing a duplication and deficiency for both the proximal and distal segments of the cluster. The Df Ll39-L62 deletes the proximal eighty 5s genes and most probably nondS DNA proximal to the cluster. The exact position of the L139 breakpoint within 56E is not known. Proximal non-5S DNA, however, is not contiguous with 5s DNA. Conversely, Df L62-L141 deletes the distal 80 genes and probably some distal non-5S DNA. The 5s genes of the L62-L141 chromosome are also not contiguous with distal non-5S DNA. The complementation of the min chromosomes, which contain about 57-69 5s genes, with the 80 genes of either deficiency chromosomes indicates that the remaining 5s genes of the deficiency chromosomes are functional. Thus the entire 5s gene cluster can be

(Oregon

R)

of 5s Genes

for Various

Number 56F Regions

DNA Hybridized (% x 10’ *SE)

Second

Chromosomes

Total Number 5s Genes

Haploid Number 5s Genes

2

54 + 2

324

162

+/Dp

L139-L141

3

6324

496

166

+/Dp

L139-L62

2.5

73 -c 6

436

+/Dp

L62-L141

2.5

72 t 4

432

subdivided into functional proximal and distal halves. This observation eiminates the possibility of a single DNA sequence outside the 5s cluster acting in cis fashion to control the transcription of the entire cluster. Such controlling elements governing a block of genes or individual genes within the cluster, however, might exist. Another redundant locus, the 18 and 28s rRNA genes of the X chromosome NO region, has also been subdivided into independently functional segments (Schalet, 1969). The restriction endonuclease results show that the entire 160 genes of the 5s cluster are bounded by Eco Rl sites with no internal sites. This physical arrangement of 5s genes was first suggested by Artavanis-Tsakonas et al. (1977). The simplest model of 5s gene organization is that all 160 genes are arranged in a single continuous cluster with no large interdispersive DNA segments not complementary to 5s rRNA. The entire cluster would yield a predicted 59 kb Eco Rl fragment similar in size to the observed fragment. The restriction pattern of the min alleles suggests a single cluster model. The saturation hybridization data demonstrate that these alleles delete about 58-65% of the 160 5s genes. Thus these alleles would give a 24-20 kb Eco Rl fragment which is in the same range as the observed min fragments of 21 kb. The possibility that wild-type 5s DNA is arranged into two discrete clusters separated by non-5S DNA which has no Eco Rl, Bg 1, Bg 11 or Barn restriction sites (Artavanis-Tsakonas et al., 1977) cannot, however, be ruled out entirely. The min deletions could simply delete this nondS DNA as well as the 55 genes. In situ hybridization studies show the 5s genes localized over at least two adjacent bands in the 56F region (Rudkin and Stollar, 1977; T. A. Grigliatti, personal communication). Although the bands of Drosophila vary in size, they contain, on the average, 3 x 104 nucleotide pairs per band (Rudkin, 1965). The amount of DNA in these two adjacent bands is sufficient to code for all 160 5s genes and yield an Eco Rl fragment of 59 kb. Judd, Shen and Kaufman (1972) propose that the band is

5s rRNA Genes 1091

Table

2. Number

of 5s Genes

for the Various

DNA Hybridized (96x10’ *SE)

min Alleles Number 5s Genes per min Allele

Total Number 5s Genes

Genotype of Chromosome 2

Number 56F Regions

minQlminQ

2

22 + 2

132

66

min’lmin’

2

19+

1

114

57

min21min2

2

23 + 1

136

69

n

wild type

minG mine

*\

min2

mm

KiZ2

DNA length (kb) l-l E

A

two adjacent bands is made up of repeating units. Indeed, one might suspect that the two adjacent bands, the 5s Eco Rl fragment and the 160 repeat units from hybridization data, are all synonymous with each other. The interband separating the two adjacent bands has a much lower relative amount of DNA and would contain either 5s genes or a small DNA sequence that does not code for 5s rRNA. In the latter case, the 160 33 genes and 59 kb Eco Rl fragment would contain a small intervening non-5S DNA segment near its midpoint. It is interesting to note that the entire cluster can be subdivided genetically into equal proximal and distal segments both of which are functional. The physical arrangement of the entire 5s gene cluster in relation to its chromosome structure is important for our understanding of chromosomal gene function and organization. Experfmentsl Drosophlls

20.7

Figure

4. Electrophoresis

of Eco Rl 5s DNA Fragments

About 5-12 pg of DNA from wild-type, min”lminO, min’lmin’ and min2/min2 flies were digested with EGO Rl restriction endonuclease. The DNA fragments were then separated on 0.4% agarose gels and transferred to nitrocellulose filters according to the method of Southern (1975). The filters were hybridized with 1251labeled 55 rRNA and autoradiographed. The sizes of linear A DNA (49 kb) and Hind Ill A fragments (H) were as described by Robinson and Landy (1977). The sizes of the Eco Rl A fragments (E) were as described by Thomas and Davis (1975). The lengths in kb were calculated assuming that the molecular weight of 1 bp is 660 daltons.

a unit of genetic function containing a single structural gene within and that the majority of its DNA is regulatory in function. In this case of 5s redundant genes, it is proposed that most of the DNA in the

Pmcedures Stocks

D. melanogaster cultures were raised at 25°C on a standard cornmeal, agar and yeast medium. A complete description of Drosophila genetic terminology is given in Lindsley and Grell (I=). The construction of segmental aneuploids for the 56Fl-9 region of chromosome 2 was described previously (Procunier and Tartof, 1975). The translocations used in this study were L139, L62 and L141 (Figure 1). Both deficiency and duplication stocks for regions spanning these translocation breakpoints were synthesized. These stable stocks were maintained asfi/fi/T(Y;P)xy/W-1 (x and y represent different translocation breakpoints). Genotypes containing various doses of the 5s genes were constructed for 5s gene number estimation (Figure 2). lsogenic Oregon R males and females have two doses of the 5s genes, one on each second chromosome.X;T(Y;P)Dp L134Ll41/+ (Dp L134 L141 designates a duplication for the region spanning the two translocation breakpoints) males have three doses of the 5s enes and were produced by mating Oregon R males to %? ,T(Y,P)Dp L134L141/SM-1 females. Since the translocation L62 breakpoint splits the 5s gene cluster into two segments (Procunier and Tartof. 1975) various genotypes were made to determine the 5s gene number of each segment. Oregon R males were mated to 2;T(Y;P)Dp Ll34L62/SM-1 and a;T(Y;P)Dp L62Ll4l/SM-1 females to yield X;T(Y;P)Dp Ll34L62/+ and X;T(Y;2)Dp L62-L141/+ male progeny, respectively, which both have approximately two and a half doses of the 5s genes. Three independently induced recessive mutants in the 5s gene cluster were isolated and designated as minO, min’ and min2. The mine allele was a spontaneous isolate from a laboratory stock, whereas both themin’ and min2 alleles were triethylenemelamineinduced (unpublished data). Flies of the genotype X;minlT(Y;S)Df Ll39L141 (Df Ll39Ll41 designates a deficiency between the translocation breakpoints and includes all the 5s genes) have a min phenotype. To estimate the number of 5s genes of the min alleles, flies homozygous for the particular min allele were made.

Temperature-Sensltlvlty

of m/n Alleles

Flies carrying the min alleles were tested for their viability and phenotypes at various tempft^res. Males homozygous for each min allele were mated to XX;Cy/T(Y;2)Df Ll34Ll41 females to yield two Fjor classes of progeny, X;min/T(Y;l)Df L139L141 males and XX/Y;Cy/min females. The progeny were reared at 16, 25 and 29”C, and the ratio of males to females was determined (Figure 3). The cultures were checked until all progeny eclosed.

Cell 1092

The control cross used Oregon R males. The min chromosomes might have a second site recessive, temperature-sensitive lethal uncovered by the Df L139-L141 chromosome. Thus min males were crossed to both Df L139-L62 and Df L62-L141 females at the various temperatures. If a lethal existed, then the male to female progeny ratio of one of these crosses would be identical to the ratio of the first cross, min males by Df L139-Ll41 females. The phenotype of flies homozygous and heterozygous for these three alleles at the different temperatures was recorded.

DNA Extraction A simple and quick method for DNA extraction using a small quantity of flies was used to estimate 5s gene numbers of various genotypes. About 1 .O g of frozen adult flies (stored at -70°C) of the appropriate genotype were homogenized at 4°C in 10 ml of a lysis solution [O.l M EDTA, 2% SDS, 0.5 M sodium perchlorate, 0.15 M NaCl (pti 8.0)] and 10 ml of water-saturated phenol (pH 8.0) chloroform (1:l v/v). l-2 vol of 1 X SSC were then added to the homogenate to the point where the viscosity was markedly reduced. The homogenate was shaken for 20 min and then centrifuged. The aqueous phase was reextracted with an equal volume of chloroform-isoamyl alcohol (24:l v/v), and the DNA was precipitated with 1 vol of cold 95% ethanol. The precipitate was dissolved in 2 ml of MUP (8 M urea, 0.24 M phosphate buffer (PB) (pH 6.8)], and the solution was passed through a hydroxyapatite (0.5 g Bio-Rad HTP in 0.24 M PB) column. The column was washed first with MUP and then with 0.014 M PB, and the DNA was eluted with 0.40 M PB. The DNA was dialyzed against 0.1 X SSC and stored at -70°C. To prepare high molecular weight DNA for restriction enzyme digestions, live adult flies were homogenized in a mortar and pestle in 10 mM MgCI,, 50 mM Naacetate, 150 mM KCI (pH 6.0) at 4°C. The homogenate was passed through three layers of cheesecloth to remove debris, and nuclei were pelleted (at 1000 x g for 5 min). The nuclei were resuspended in 0.1 M NaCl and 25 mM EDTA and lysed by the adding SDS to l%, and the mixture was phenol-chloroform-extracted. Following an additional phenolchloroform extraction, the DNA was ethanol-precipitated and dissolved in buffer TNE [lo mM Tris, 10 mM NaCl, 1 mM EDTA (pH 7.5)]. A saturated CsCl solution (technical grade; Kawecki Berylco Industries) was added to the DNA solution until a final concentration of 1.70 g/cc was obtained. The DNA was banded by centrifugation (30,008 rpm in an SW40 Spinco rotor at 20°C for 72 hr). located by optical density, precipitated with ethanol and dissolved in TNE buffer.

5S rRNA

Preparatlon

and Labeling

Frozen wild-type adult flies (-70°C) were homogenized in equal volumes of phenol-chloroform and 0.14 M Na acetate (pH 4.5) buffer. After centrifugation. the aqueous phase was reextracted with phenol-chloroform and centrifuged, and the aqueous phase was applied to a DEAE-cellulose column equilibrated with buffer N [0.014 M Na acetate, 0.3 M NaCl (pH 4.5)]. The column was washed with buffer N and RNA was eluted with 0.14 M Na acetate, 1 .I M NaCI. (pH 4.5). The RNA was precipitated with ethanol and dissolved in 0.01 M Na acetate, 0.1 M NaCl, 1 mM EDTA (pH 6.0) at a concentration of 1.0-I .5 mg/ml. High molecular weight RNA was precipitated overnight at 4°C by adding solid NaCl to 2.0 M and then removed by centrifugation. The 4 and 5s RNA was precipitated with ethanol, collected and dissolved in Sephadex buffer [0.75 M NaCl. 1% methanol, 0.05 M acetic acid (pH 5.0)]. The RNA was loaded onto a 195X 2.5 cm Sephadex G-108 column to separate the 5s rRNA from the remaining 16 and 265 rRNA and 4s RNA. Fractions containing 5s RNA were pooled, and the RNA was ethanol-precipitated and dissolved in RPC-5 buffer [O.Ol M Na acetate, 0.01 M MgCI,, 0.45 M NaCl (pH 4.5)]. The RNA was applied to an RPC-5 column (0.9 x 60 cm), eluted at 14 ml/hr with a 200 ml gradient (0.525-0.7 M NaCI) and collected. The 5s RNA was iodinated in vitro by the method of Grigliatti et al. (1974) to a specific activity of about 2.4 x lO’cpm/pg. Unlabeled 16 and 285

rRNAs were

DNA-RNA

prepared

by the method

of Tartof

and Perry

(1970).

Hybridization

The hybridization reaction was performed by a method developed by A. Delaney (personal communication). DNA in 0.1 X SSC was denatured by heating for 5 min at 100°C and quickly cooled on ice. The 1Z51-labeled 5s rRNA hybridization reactions were carried out for 30 min at 65°C in 0.66 ml of hybridization buffer [0.15 M NaCI, 0.015 M Na acetate, 20 mM EDTA (pH 5.0)) containing about 50 pg of denatured DNA and 5s RNA at saturation conditions (1 pg/ml). At the end of the reaction time, the hybridization solution was loaded onto an agarose column (A = 5m; 0.70 x 50 cm; equilibrated with hybridization buffer without EDTA at 85°C) to separate the DNA and hybrid RNA-DNA from the unhybridized 5s rRNA. In the exclusion volume, the amount of DNA was estimated by absorbance at 260 nm and the hybridized 5s rRNA by radioactivity. The number of Y-labeled 5s rRNA counts for 50 rg of DNA was about 65,ooO cpm, and background values (hybridization solutions containing no DNA) were c 50 cpm. Preincubation of denatured DNA in the hybridization solution at 65°C for 2 hr and subsequent hybridization showed no change in the amount of hybridized 5s rRNA, indicating negligible DNA renaturation under these conditions. The hybridization values for each particular genotype were averaged and the standard error (SE) was calculated. The number of 5s genes was determined using the molecular weights of the diploid genome of D. melanogaster and 5s rRNA as 2.4 x 10” and 4.0 x IO’, respectively (Tartof and Perry, 1970).

Restriction

Enzyme

Dlgestbns

Endonucleases Eco Rl and Hind Ill were obtained from Miles Laboratories. Approximately 5-12 pg of high molecular weight Drosophila DNA were digested with Eco Rl in 200 ~1 of 0.05 M Tris-HCI (pH 7.5, 5 mM MgCI,, 0.06 M NaCl, 1 mM dithiothreitol and 0.1 mg/ml gelatin at an enzyme concentration of 0.5 units of enzyme per pg of DNA for 12 hr at 37°C. After digestion, the reaction mixture was made 2 mM EDTA and 0.2 M Na acetate. The DNA was precipitated with 3 vol of ethanol at -20°C for 8 hr. collected by centrifugation and dissolved in electrophoresis buffer.

Agarose

Gel Electrophoresis

and Filter Hybridizatbn

Electrophoresis was carried out on a 15 x 50 x 0.5 cm horizontal slab gel of 0.4% agarose (Seakem) run on a water-cooled Shandon apparatus. Samples (40 ~1) containing from 5-12 pg of DNA in electrophoresis buffer [40 mM Tris-phosphate (pH 8.0), 20 mM NaH,PO,, 1 mM EDTA] were made 0.1% SDS, 0.1% bromophenol blue and 10% sucrose. The samples were loaded into the wells and sealed with Vaseline. Electrophoresis was for 42 hr at 10 mA. The gelswere calibrated by running Hind Ill (Robinson and Landy, 1977) and Eco RI (Thomas and Davis, 1975) digests of A DNA in parallel. Following electrophoresis. the Eco RI DNA fragments were transferred to nitrocellulose filters as described by Southern (1975). Hybridization of the filters was carried out for 3 hr at 65°C in 2 x SSC containing 0.5% SDS, 2.5 nfvl 1251-labeled 5s rRNA (4.0 x IO7 cpm/pg) and 18 plus 285 rRNA (8 mg/ml). After hybridization, the filters were washed twice in 250 ml of 2 x SSC at 65°C for 10 min and treated with RNAase (10 mg/ml; Worthington) in 2 x SSC for 1 hr at 37°C. The filters were then washed twice with 2 x SSC containing 0.1% SDS and air-dried. Autoradiography was for 24 hr at -70°C using Kodak X-Omat-R film and a DuPont Lighting Plus intensifying screen.

Acknowledgments This work was supported by a National Research Council of Canada grant, a National Cancer Institute of Canada contract to D. T. Suzuki and a Medical Research Council of Canada grant to G. M. Tener. The authors wish to thank Drs. Suzuki and Tener

55 rRNA 1093

Genes

for their advice and the use of their facilities. The mutants were isolated in the laboratory of Dr. Ken Tartof, whose assistance is appreciated. Special thanks to Drs. Tom Grigliatti, Ian Gillam and Allan Delaney for their expert technical assistance and comments. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact. Received

May 6,1978;

revised

August

2.1978

References Artavanis-Tsakonas, S., Schedl, C. T., Pirrotta. and Gehring, W. J. (1977). Cell 72, 1057-1067. Grigliatti, T. A., White, 6. N., Tener. Suzuki, D. T. (1974). Proc. Nat. Acad. Hershey, Davidson,

V.. Steward,

R.

G. M., Kaufman, T. C. and Sci. USA 77, 3527-3531.

N. D., Conrad, S. E., Sodja, A., Yen, P. H., Cohen, M., N., llgen, C. and Carbon, J. (1977). Cell 17, 585-598.

Judd, B. H., Shen. 139-156.

M. W. and Kaufman,

Lindsley, D. L. and Grell, Publication 627.

T. C. (1972).

E. H. (1966).

Carnegie

Genetics

71,

Inst. Washington

Lindsley. D. L., Sandler, L., Baker, 6. S.. Carpenter, A. T. C., Denell, R. E., Hall, J. C.. Jacobs, P. A., Miklos, G. L. G., Davis, 8. K., Gethamn. R. C., Hardy, R. W., Hessler, A., Miller, S. M., Nozawa, H., Parry, D. M. and Gould-Somero, M. (1972). Genetics 71. 157-184. Miller,

L. and Knowland,

J. (1972).

Biochem.

Genet.

Procunier,

J. D. and Tartof,

K. D. (1975).

Genetics87,

Procunier,

J. D. and Tartof.

K. D. (1976).

Nature263,

Procunier, 209.

J. D. and Williamson,

Ritossa, F. M.. Atwood, 54, 663-676. Robinson,

L. H. and Landy, G. T. (1965).

Rudkin,

G. T. and Stellar,

Schalet,

A. (1969).

A. (1977).

Genetics52,

Scheer, U., Trendelenburg, Cell Biol. 69, 465-489. Shermoen, Tartof, Thomas,

K. D.and

Genetics

I-31.

Nature265,472-473.

133-153.

B. I. (1975).

J. Mol. Biol.98,

Franke.

W. W. (1976).

Cell 4, 275-280.

Perry,

R. P. (1970).

J. Mol.

R. W. (1975).

J. Mol. Biol. 91, 315-328.

L. and Berger,

Wimber.

D. E. and

E. (1978). Steffensen,

J.

503-517.

M. and Davis,

Weber, 641.

S. (1966).

Gene2,

M. F. and

A. W. and Kiefer, E. M. (1975).

Biol. 39, 198-

665-681.

B. D. (1977).

Genetics63,

515-523. 255-257.

Dev.

K. C. and Spiegelman,

Rudkin,

Southern,

J. H. (1974).

6,65-73.

Biol.57,

171-183.

Biochemistry

15, 5511-5519.

D. M. (1970).

Science

770, 639-