Gene linkage by RNA-DNA hybridization

J. Mol. Biol. (1968) 34, 661-680

Gene Linkage by RNA-DNA

Hybridization

I. Unique DNA Sequences Homologous to 4 s RNA, 5 s RNA and Ribosomal RNA DONALD D. BROWN AND CARL S. WEBERt

Department of Embryology Carnegie Institution of Washington, 115 West University Baltimore, Mayland 21210, U.X.A.

Parkway

(Received 8 Januury 1968) A method for measuring sequence homology between high specific activity RNA’s and DNA of Xenopua Iaevia is described. A CsCl centrifugation step fractionates the DNA according to its buoyant density. The DNA in each fraction is denatured, immobilized on a nitrocellulose filter, and then hybridized with P-RNA. The technique combines specificity and versatility not afforded by other hybridktion methods. Specificity arises from the fact that the DNA’s homologous to different classes of RNA band at unique buoyant densities. Essentially all DNA homologous to 28 s and 18 s ribosomal RNA (rDNA) bands as a heavy satellite. The DNA homologous to 5 s RNA bands to the light side of the bulk DNA, and DNA homologous to 4 s RNA is distributed throughout the band of the bulk DNA and to the heavy side. Since the DNA’s homologous to each of these RNA classes are separated by C&l fractionation, competition experiments have increased specikity and have been used to demonstrate that the DNA’s do not share common nucleotide sequences. The high-density rDNA comprises about 0.0S7°h of the genome as determined by hybridization with saturating amounts of rRNA; this amounts to about 460 of the 28 s and 18 s genes for each haploid complement of chromosomes. This rDNA is clustered on a single chromosome, since more than 99% of it is lost by a deletion (the nucleolar mutation) which segregates as a simple Mendelian factor. This deletion does not affect the DNA homologous to 4 s and 5 s RNA, supporting the observation that these DNA sequences are not intermingled with the rDNA. Since a linear relationship exists between the amount of DNA immobilized on filters and the extent of hybridization with the three groups of [3H]RNA, the technique can be used to measure the amount of homologous sequences in unknown DNA preparations even when the RNA is used in subsaturating amounts. The relative abundance of nucleotide sequences homologous to rRNA and 6 s RNA in X. Latvia liver, erythrocyte, and embryonic DNA has been found to be similar.

1. Introduction This paper describes a highly specific and versatile method for detecting the DNA which is homologous to ribosomal RNA, 5 s RNA and 4 s RNA in the genome of Xenopw Levis, the South African clawed toad. The method couples a preliminary fractionation of the DNA according to its buoyant density by centrifugation in C&l (Wallace & Birnstiel, 1966) with hybridization of RNA to DNA immobilized on nitrocellulose flters (Gillespie & Spiegelman, 1965). t Present address: Department Baltimore, Md., U.S.A.

of Biology,

University

661

of Maryland,

Baltimore

County,

662

D. D. BROWN

AND

C. S. WEBER

The preliminary CsCI fractionation of the DNA increases tile xpecificity of the subsequent hybridization reaction. An example of the kind of information gained from this technique is the finding that DNA homologous to 28 s and 18 s RNA can be separated from DNA homologous to 4 s RNA and 5 s RNA. In addition, competition experiments using CsCl-fractionated DNA demonstrate the specific competition only at homologous loci, as evidenced by the reduction of RNA counts binding to DNA of a certain buoyant density. Finally, the technique can be used to quantitate the amount of DNA homologous to these three classes of RNA. This is possible for a wide range of [3H]RNA concentrations, since the amount of RNA which is hybridized is related linearly to the amount of homologous DNA on a filter.

2. Materials and Methods (a) Culture of radioactive cells for RNA isolation Radioactive RNA was pursed from [3H]uridine-labeled cells derived from adult X. bevis kidney. The tissue was minced with scissors and dissociated with a mixture of trypsin, pancreatin and collagenase using standard dispersal techniques. A l-ml. suspension containing approximately lo6 cells was put into a Falcon plastic tissue culture flask (75 cma surface) containing 15 ml. of growth medium. The nutrient medium, Waymouth’s MB 752/l (Grand Island Biological Company) diluted with 0.64 vol. of water, was supplemented with 10% fetal calf serum, penicillin and streptomycin. The cultures were gassed with 5% CO2 in air; this atmosphere maintained a pH of 7.1 to 7.3. Culture flasks were incubated at 23 to 25°C in the dark. After 3 days, all unattached cells were removed and fresh medium was added. Cultures were fed every 4 to 5 days thereafter by a complete exchange of medium. Twelve days after seeding, the monolayer was confluent (approximately 8 x lo* cells/cm2), and the cells were trypsinized and split 1: 2. At about 12-day intervals the cells were serially subcultured. The primary culture contained a mixture of epithelioid and fibroblastic cells with the latter predominating after the first few subcultures. The [5-3H]uridine (10 c/m-mole, from Nuclear Chicago Corp.) was added to the sixth subculture at a concentration of 10 &ml. when the monolayer was about one-third confluent. After the cells had doubled at least twice in the presence of isotope (a period of about 4 days), they were “chased” in non-radioactive medium for 8 hr, trypsinized and collected. The washed cells were frozen at -70°C until they were processed. (b) Preparation of radioactive RNA 3H-labeled rRNAt, 6 s RNA and 4 s RNA were puri6ed from the same preparation of radioactive tissue culture cells. The frozen cells were homogenized in RSB medium and immediately centrifuged at 100,000 g for 1 hr. The supernatant fraction was collected for 3H-labeled 4 s RNA isolation; 3H-labeled rRNA and 3H-labeled 5 s RNA were extracted from the ribosomes in the pellet. (i) Pur$kation of 3H-labded 4 s RNA The 3H-labeled 4 s RNA was purified from the high-speed supernatant fraction by cold phenol extraction (without sodium lauryl sulfate) and subsequent adsorption to and elution from a DEAE cellulose column. The fractions containing 4 s RNA were pooled, dialyzed, and rechromatographed either on methylated albumin-kieselguhr or protamineCelite columns as described previously (Brown & Littna, 1966). One important modification was the incorporation of a layer of Celite below the MAK or protamine-Celite in each t Abbreviations used: rRNA, 28 s and 18 s ribosomal RNA; rDNA, the DNA homologous to 28 s and 18 s ribosomal RNA by molecular hybridization; 4 s DNA and 5 s DNA, the DNA homologous to 4 s RNA and 6 s RNA, respectively; RSB, reticulocyte standard buffer, which

contains 0.01 M-Tris, 0.01 an-KCl, 0.0015 &i-MgCl,, at pH 7.4; SSC is 0.16 M-NaCl and 0.016 Msodium citrate; MAK, methylated albumin-kieselguhr; 0-nzl embryos of Xenopm Zaevis are homozygous for the anucleolate mutation; l-w and 2-nu embryos are haterozygotes and wild type, respectively (Elsdale, Fischberg & Smith, 1968).

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column 8s origirmlly described by Mandell & Hershey (1960); this Celite pad traps basic proteins released from the column which otherwise would interfere with the subsequent hybridization reaction. Finally, the RNA wss dialyzed against distilled water, concentrated in VCLCUO to 1 to 10 ,.&ml., and then slowly filtered through a washed Millipore HA filter. The RNA was assayed for optic81 density rend acid-insoluble radioactive material 8nd stored at -70°C. (ii) Puri@ztiort of aH-labeled TRNA In the experiments reported in this paper, 28 s and 18 s rRNA were purified together, and will be collectively referred to as “rRNA”, in contrast with 5 s and 4 s RNA. The high-speed pellet which contained the ribosomes was extmcted with 0.1 ~-sodium acetate (pH 6) containing 4 pg polyvinyl sulf8te/ml. 8nd 0.5% sodium lauryl sulf8te. The suspension was shaken with phenol at 0°C snd the aqueous phase was passed directly over a 30-cm Sephadex GlOO column. The unretarded volume containing the rRNA w8s collected separately from the retarded peak which contained 5 s and some ribosomebound 4 s RNA (Brown & Littn8, 1966). The unretarded volume from Sephadex ~8s treated with DNase (10 pg/ml. electrophoretically pure) in 3 mM-MgCl, for 10 min at room temperature and the RNA was adsorbed to and eluted from 8 2 cm x 2 cm MAK column over Celite with a gradient of N8Cl from 02 M to 1.2 M. The rRNA elutes as a single peak at about 0.8 M-NaCl. The preparation w8s dialyzed against distilled wetter, concentrated to 10 to 50 pg/ml., filtered through a washed Millipore HA filter, and stored at -70°C. Since there is no preferential loss of 28 s or 18 s during this treatment, rRNA preparations contain equimolar 8mounts of 28 s and 18 s rRNA. The s8me is true for non-radioactive rRNA preparations (see Materials and Methods, section (c) ). (iii) Preparation of SH-labeled 5 s RNA The retarded volume from the Sephadex fractioncltion was concentrated in vacua and the 5 s RNA was sep8rated from 4 s RNA and traces of rRNA on a 6-ft Sephadex GlOO column (Galibert, Larsen, Lelong & Boiron, 1965; Brown & Littna, 1966). The 5 s RNA was purified further by fractionation on 8 protamine-Celite column (Brown & Littna, 1966), and then dialyzed, concentrated and tiltered 8s described above for the other RNA%. The final specific activities of the 3H-18beled RNA preparations used in these experiments were 4.0 x 105 cts/min/~g RNA. The radioactivity on Millipore filters was counted in 8 liquid-scintillation spectrometer. (c) Preparadion of non-ra&oactive rRNA, 5 s RNA and 4 s RNA Non-radioactive rRNA and 6 s RNA were extracted from ribosomes which had been isolated from unfertilized eggs of X. Zaevti ; 4 s RNA was purified from the high-speed supernatant fraction of 8dult X. kzevis liver. The techniques for preparation of cold rRNA and 6 s RNA were the same 8s those used for their radioactive counterparts, but with an additional centrifugation at 16,000 rev./mm for 1Omin just prior to the high-speed centrifugation. The unfertilized egg ribosomes which remain in the postmitochondrial supernatant fraction following this low-speed centrifugation are predominantly monosomes with very little bound 4 s RNA and presumably very small amounts of associated messenger RNA (Brown, 1965; Brown & Littna, 1966). The centrifug8tion steps enrich the high-speed pellet for 5 s RNA relative to 4 s RNA and for rRNA relative to DNA-like RNA since the latter is partially degraded in neutr81 homogenates (Brown & Littna, 1964). (d) Prepar&m of DNA In most of the experiments reported here, X. lae& DNA was prepared from nucle8ted erythrocytes. The erythrocytes were collected in 1 x SSC, washed twice, and then lysed by suspension in distilled water at 0°C for 10 min. The nuclei were pelleted at 6000 g for 10 min. The pellet was dispersed in 1 x SSC with gentle homogeniz8tion, and pronase and sodium lauryl sulfate were added to final concentrations of 1 mg/ml. and 0*6’$& respectively. Pronase digestion (Berms & Thomas, 1966) was carried out for 4 to 6 hr at 37’C. An equal volume of phenol was sdded and the suspension gently rotated overnight

664

D. D. BROWN

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at 4°C. The remainder of the purification was effected by standard methods involving RNase and amyhe digestion, winding the DNA out of ethanol or isopropanol, and several extractions with phenol to remove any remaining protein (Marmur, 1961; Dawid, 1965). Erythrocytes from ten adult X. Zuevia yielded about 20 mg of high-molecular weight DNA (estimated at greater than 5 x IOr daltons). DNA from homozygous anucleolate (O-mu) embryos of X. Zuevis (Elsdalo, Fischberg & Smith, 1958) was prepared by homogenizing stage 40 (Nieuwkoop & Faber, 1956) swimming embryos in 0.15 u-NaCl, 0.1 M-EDTA (pH 8); 0.5% sodium lauryl sulfate and an equal volume of phenol was added, and the samples were allowed to rotate gently overnight at 4°C. The aqueous phase was collected and dialyzed against 0.1 x SSC until the last traces of phenol were gone, and then the solution was made 1 x SSC and treated with 20 pg of RNflee/ml. for about 1 hr at room temperature. The resulting extract was adsorbed directly onto a 2 cm x 2 cm MAK column (over Celite) and washed with 0.2 M-NaCl-0.05 rd-Tris (pH 7.2) until no more ultraviolet-absorbing material was eluted. The DNA was eluted with 08 M-Nacl-0.05 M-T& (pH ‘7.2) and then dialyzed against 0~1x SSC, concentrated in vocuo, and stored at 4°C with a small amount of chloroform. The final yield of DNA from O-VU embryos was 05 to 1.0 pg DNA/embryo. (e) CsCl centrifugation and DNA immobilization Native or denatured DNA was centrifuged in a Spinco preparative ultracentrifuge in either a type 65 fixed-angle rotor or a type SW39 horizontal rotor. Centrifugation in the fixed-angle rotor producea a shallower non-linear C&l gradient than does the horizontal rotor (Flamm, Bond & Burr, 1966) and therefore effects a greater separation of the rDNA satellite from the bulk DNA. In both cases centrifugation was performed at 33,000 rev./min for 48 to ‘70 hr at 25°C. After centrifugation, the content of each tube was fractionated by drop collection and the optical density at 260 rnp of each fraction was measured. Each fraction W&B made 0.1 N with KOH to denature the DNA, and then neutralized and diluted to about 8 ml. with 4 x SSC. Millipore HA grid filters cut to fit loosely in scintillation vials (2 cm diameter) were stamped out of sheets and soaked in 4 x SSC for at least 24 hr before use. Each fraction from the C&l gradient W&EIpassed at about 1 to 2 ml&in through a soaked Millipore filter with its grid side up. The filters were blotted, heated overnight at 70°C in an open air oven, and then stored in closed vials at room temperature or used directly for hybridization. (f) Hybridization reaction As many &8 100 whole filters or 200 half-filters were hybridized together in 4 x SSC in a 2.5cm diameter plastic vial. The volume required wm 2 ml. for one to ten whole filters, and 1 ml. for each additional ten filters. Blank filters without DNA were interspersed throughout the stack of DNA-containing filters. The complete hybridization medium was mixed immediately beforehand and then added to the stack of dry filters. After carefully removing all the air bubbles trapped between filters, the vial was closed and incubated at 70 to 72°C for 15 to 20 hr. At the end of the hybridization period, the stack of filters was rinsed several times with 2 x SSC and treated with DNase-free RNase (50 pg/ml.) in 2 x SSC with gentle shaking for 1 hr at room temperature. The filters were washed again several times with 2 x SSC. Then each filter was placed individually in about 20 ml. of 2 x SSC and soaked for 2 to 3 hr, after which it was placed on a flitted glass filter holder. Each side of the filter was washed by passing 5 to 10 ml. of 2 x SSC through the filter. When half-flltere were used, the 6nal washing step on the filter holder was omitted. The filters were then blotted grid surface up on filter paper and dried for several hours at 37°C. After a filter had been counted, it was assayed for the amount of DNA it contained by acid hydrolysis. The alter was washed free of the fluor (toluene base) with chloroform, dried and hydrolyzed in 1-O N-HCl for 15 min at 100°C. The optical density at 260 rnp of 1 mg hydrolyzed DNA/ml. in 1.0 N-HCl is 27.8. Whenever DNA wa prefractionated in C&l and then immobilized on alters, there was no cross-contamination between adjacent filters during the overnight hybridization period. However, DNA-titers which had been prepared from unfractionated DNA occasionally lost their DNA during the hybridization step. This was detectable not only by loss of

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BY HYBRIDIZATION

DNA from a filter, but also by the increased radioactivity of blank filters which were directly adjacent to the defective DNA-filter in the stack. In most experiments data have been plotted without background correction. In a typical hybridization reaction, filters were hybridized in 5 ml. of 4~ SSC containing 2 pg eH-labeled rRNA (about 8 x lo6 cts/min). Following hybridization, RNase digestion and the washing procedures, blank whole filters retained from 60 to 100 cts/min (less than O-Olo/o of added counts); blank half filters retain 25 to 60 cts/miu eaoh. Blank values for 3H-labeled 4 s and 3H-labeled 6 s RNA hybridization under comparable conditions were approximately half and one-fourth the levels of 3H-labeled rRNA blanks, respectively.

3. Results (a) Dependence of rDNA separation on DNA concentration Resolution of the ribosomal DNA satellite by CsCl centrifugation depends upon both the molecular weight and the concentration of the DNA. There is an inverse relationship between the molecular weight of the DNA and the DNA concentration which shows maximum satellite separation (Fig. 1). As much as 100 pg of sheared td)

-600

t

- 400

e)

I!-

-200 0 I 1200 _ .E -800

5 Y

-400

2.? t; .%

,o f) Rn

CT

Tube no.

Fro. 1. Effect of DNA concentration and molecular weight on resolution of the rDNA satellite by C&l centrifugation. Native DNA, undegraded ((a), (b), (c)) and sheared ((a), (e), (f)), was centrifuged to equilibrium in a fixed angle rotor. The three ooncentrations of DNA in a final volume of 45 ml. (p = 1.730) were 108 cl~ ((a) and (d)), 198 H ((b) and (e)) and 396 H ((c) and (f)). Identioal portions were taken from each fraction for hybridization with [sH]rRNA. All the filters prepared from the six gradients were hybridized together in a single vial containing 13 ccg[sH@RNA in 8 ml. of 4 x SSC; blank 6.lters were present to monitor background (40 cts/min). Estimated molecular weights for the two DNA preparations were 6 X 10 and 6 to ‘7x 106, respeatively, for undegraded and sheared Optioal density at 260 mp; -@-a--, radioactivity in DNA (Brown & Weber, 1968). -, cta/min. The arrow denotes the position of the rDNA satellite in an analogous experiment with 25 pg of the same DNA preparation.

D.

666

D. BROWN

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C. S. WEBER

native DNA (about 6 to 7 x lo6 daltons) can be centrifuged in 4.5 ml. of CsCl solution in a 65 fixed-angle rotor with good resolution of the rDNA satellite. However, its resolution in undegraded native DNA is impaired even at 100 pg DNA/ml. (Fig. 1). The poor separation of the rDNA satellite from high molecular weight DNA is not caused by a true change in its buoyant density as inferred by the experiments of Birnstiel, Wallace, Sirlin & Fischberg (1966; Birnstiel, 1967). It is a concentrationdependent effect, since at a concentration of 25 pg of high molecular weight DNA/ml., the rDNA component separated from the bulk DNA by the same distance as in sheared DNA. (b) Separation of DNA humdogoua to 58, 4s and rRNA by Cd71fractionation The sequences complementary to 5 s, 4 s and rRNA are located on DNA fragments which differ in buoyant density. Whereas rRNA hybridizes with DNA of high buoyant density, 5 s RNA hybridizes with DNA which is lighter than bulk DNA (Fig. 2). In contrast, 4 s RNA hybridizes with DNA fragments which are distributed throughout the main DNA peak as well as to its heavy side (Fig. 3). Despite these specific distribution patterns, there is some overlap in the hybidization. For example, some

-t so

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04

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I.E 123 h .e 100 ‘i-Q .P 22 .oo )

-i !OO

0.1

?s d d

(

02

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-. C Tube no.

FIG. 2. Hybridization of C&l-fraotion&ed native and denatured DNA with 3H-l&eled rRNA and 3H-labeled 6 s RNA. About 60 pg of sheared native DNA (6 to 7 x lOa dsltona) and 60 pg of the same DNA after alkali denatoration and reneutralization were fractionated in Cscl in a fixed angle rotor (p = 1.710). Each fraction wea rtlkfdi denatured and the DNA immobilized on a filter. Each filter was split and sets of half-filters were hybridized with either 1.8 a [3H]rRNA in 4 x SSC (--O--O-) or with O-2 pg 3H-labeled 6 s RNA in 4 ml. of 4 x SSC (--O--O --). The results are plotted without background oorreotion. Upper Figure (a) is native DNA; lower Figure (b) is denatured DNA: . optical density at 260 mp.

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hybridization of the 3H-labeled rRNA preparation occurs with the bulk DNA (Figs 1 to 3), and some 3H-labeled 5 s RNA hybridizes in the rDNA satellite region (Fig. 2). This overlap is not due to the presence of homologous sequences in DNA fragments of different densities but rather to contamination of the [3H]RNA preparations. This distinction has been resolved by competition experiments which will be described in the following sections. (a)

(b)

'3 :: .o -0

z d 6 0.4-

80--800

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Tube no.

3. Hybridization of CsCl-fractionated native and denatured DNA with [3H]rRNA and 3H-labeled 4 s RNA. About 50 pg of sheared native DNA (6 to 7 x lo6 daltons) and 50 pg of the same DNA after alkali denaturation and reneutralization were fractionated in C&l in a fixed angle rotor. Filters were prepared for each fraction and split. Sets of half-filters were hybridized with either 0.9 pg [3H]rRNA in 4 ml. of 4 x SSC (-@-a-) or with 1.2 pg of 3H-labeled 4 s RNA in the same volume (- - ()- -O- -). The results are plotted without background correction. Upper Figure (a) is native DNA; lower Figure (b) is denatured DNA; -, optical density at 260 mp. FIG.

(c) CornSpetition between non-radiocxtive rRNA

and 3H-labeled rRNA

The 3H-labeled rRNA hybridizes with CsCl-fractionated DNA molecules which are distributed in two regions of the CsCl gradient, namely in t,he high-density satellite and bulk DNA regions. In the experiments recorded in Figures 2 and 3, about 80% of the hybridized 3H-labeled rRNA was bound to DNA in the satellite region, while the remainder was bound to the bulk DNA. Two explanations are possible for this observation: either both of these populations of DNA molecules have sequences which are homologous with rRNA, or other classes of radioactive RNA, such as DNA-like RNA, are present as trace contaminants in the labeled rRNA preparation. These alternatives were tested by a competition experiment in which non-radioactive rRNA was added as a competitor to the 3H-labeled rRNA. Since the competing unlabeled rRNA was isolated from the same animal but from a different tissue, it was hoped that t,he unlabeled DNA-like RNA molecules would be different 44

D. D. BROWN

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from those in the 3H-labeled rRNA preparation. In addition, the cold rRNA was extracted from unfertilized egg monosomes which are largely “unprogrammed” and presumably very low in DNA-like RNA content (Brown, 1965). A competition experiment (Fig. 4) was used both to investigate the hybridization with bulk DNA and to demonstrate the absence of DNA homologous to rRNA in the anucleolate (0-nu) mutant embryos of X. Zaewis.Wallace & Birnstiel (1966) reported that no more I

Control

(2-m)

I

Anucleolate (0 -nu)

1

Tube no. FIG. 4. Hybridization of [3H]rRNA with wild-type and 0-no DNA fractionated in CsCl. 50 pg of X. .?uev& wild-type erythrocyte DNA and an equal amount of 0-nu DNA were fractionated in CsCl in a fixed angle rotor. Then each filter was split and one set of half-filters was hybridized in 5 ml. of 4 x SSC containing 1.8 pg [3H]rRNA (-e-m--); the other set was hybridized with the same amount of [3H]rRNA plus 10 pg of cold rRNA (--O--O--). -O--O-, Optical density at 260 mp. The lower plot of O-nu DNA has an enlarged ots/min scale to magnify the relatively low level of binding with the bulk DNA. Background filter radioactivity has been subtracted.

than 5% of the rDNA level found in wild-type DNA could be present in 0-nu embryos; the experiment recorded in Figure 4 confirms their findings, but with increased sensitivity. The 0-w and wild-type DNA were CsCl-fractionated, and DNA from each fraction of the gradient was immobilized on a filter. Each filter was split and one set of half-alters was hybridized with 3H-labeled rRNA alone while the other set was hybridized with an identical portion of 3H-labeled rRNA admixed with cold rRNA (Fig. 4). There is no detectable hybridization above background in the satellite region of 0-nu DNA, but a small number of counts have bound to the bulk DNA. This count level is about the same as that which bound to the bulk of wild-type (2-nu) DNA. Whereas competition with cold rRNA reduced the hybridization of the 3H-labeled rRNA with wild-type DNA in the satellite region, there was no reduction of the [3H]RNA that hybridized with the bulk of either 0-nu or 2-nu DNA. This finding demonstrates that the low level of radioactive material which binds with the bulk DNA does not reflect the presence of sequences homologous with rRNA. The sensitivity of this experiment was such that it would have detected as little as 1% of the level of rDNA present in wild-type (2-m) DNA.

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of 5s RNA

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and 4s RNA

with 3H-kzbeEed rRNA

CsCl fractionation of DNA before hybridization enhances the specificity of crosscompetition experiments using 5 S, 4 s and rRNA. In these experiments the rDNA was separated further from the bulk DNA by its hybridization with rRNA before centrifugation (Wallace & Birnstiel, 1966). This technique is described in detail in the accompanying paper (Brown & Weber, 1968). Following CsCl-fractionation, the rRNA which had been bound to rDNA was released by the alkali denaturation step and then discarded into the filtrate when the DNA was immobilized. The filters were split and hybridized with either 3H-labeled rRNA, 3H-labeled 5 s RNA or 3H-labeled 4 s RNA in the presence or absence of different cold RNA preparations as competitors. The results from cross-competition experiments between 5 s and rRNA are shown in Figure 5. A 50-fold excess of cold rRNA which competes for more than

No

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5~ RNA

competition

competition

i I

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FIU. 5. Cross-competition experiment between rRNA and 5s RNA using CsCl-fractionated DNA. About 70 pg of DNA were alkali denatured, hybridized with 30 pg of cold rRNA, divided into three parts and centrifuged to equilibrium in the SW39 rotor. This prehybridization increases the buoyant density of rDNA and thus separates it even further from the bulk DNA (Wallace & Birnstiel, 1966; Brown & Weber, 1968). Each of the three sets of DNA filters was split and halfsets were hybridized with either 0.2 pg [sH]rRNA (-a-@-) or 0.1 pg sH-labeled 5 s RNA without competitor RNA (left), the second set was (--m--a--). One set was hybridized hybridized in the presence of 10 pg of cold rRNA (middle), and the third set was hybridized in the presence of 10 pg of cold 5 s RNA (right). -O--O--, Optical density at 200 rnp.

90% of 3H-labeled rRNA hybridization reduces 3H-labeled 5 s RNA binding by about 40% in this experiment. There may be contamination of this cold rRNA preparation with a trace of cold 5 s RNA. In other experiments (see Table 1) several hundredfold excess of a different preparation of cold rRNA caused about 40% reduction in 3H-labeled 5 s RNA hybridization with its homologous low-density DNA. However, the previous competition experiment (Fig. 4) failed to demonstrate any sequence homology of rRNA with DNA of a buoyant density as light as the DNA homologous to 5 s RNA. Another possible explanation for this cross competition is a non-specific inhibition of hybridization caused by the large amounts of unlabeled high-molecular weight rRNA. This alternative has not been explored in detail. However, in the reverse competition experiments, a lOO-fold excess of cold low-molecular weight 5 s RNA did not reduce 3H-labeled rRNA hybridization, although it reduces 3H-labeled 5 s RNA hybridization by more than 90%. Similar

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results were obtained for the cross-competition experiments between 4 s RNA and rRNA as shown in Figure 6. Finally, cross competition of 4 s RNA and 5 s RNA demonstrates the absence of common nucleotide sequences (Fig. 7). Cold 5 s RNA competes for some binding of the 3H-labeled 4 s RNA preparation on the light side of the DNA at the density of 5 s DNA, suggesting that some 3H-labeled 5 s RNA contaminated the 3H-labeled 4 s RNA preparation. Therefore, we conclude that t’he DNA’s which are homologous with these three clan+ of RN14 can be distinguished by their buoyant density and, at the level of sensitivity presented here, do not have sequence homologies in common. No

rRNA

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competition

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competltion

., I r

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$ 0.20 s d 6 O.I0 -c-a IO Tube

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FIQ. 6. Cross-competition experiments between rRNA and 4 sRNA using CsCl-fractionated DNA. The conditions were identical to those of the experiment shown in Fig. 5 except for the hybridization conditions. Each half-set of filters was hybridized with either 0.2 pg [3H]rRNA (-@-a--), or 0.3 pg sH-labeled 4 s RNA (-- l -- l --). One set was hybridized without competitor RNA (left), the second set was hybridized in the presence of 5 pg of cold rRNA (middle), and the third set hybridized in the presence of 5 pg of 4 s RNA (right). -O-G-, Optical density at 260 mp.

3H-lobeled

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20 f2 N 6 00

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FIQ. 7. Competition of cold 5 s and 4 s RNA for hybridization of 3H-labeled 4s and 3H-labeled 5 s RNA. Denatured DNA was fractionated in CsCl in two separate tubes in an angle rotor. The filters were prepared and split. One set of filters was hybridized with 3.6 pg 3H-labeled 4 s RNA (left) in the presence (--O--O--) or absence (-•-•--) of 4 pg cold 5 s RNA. The other set was hybridized with 0.5 pg 3H-labeled 5 s RNA (right) in the presence (--O--O-) or absence (--a-e-) of 6 pg of cold 4 s RNA. -, Optical density at 260 mp.

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Although most of the DNA which is homologous to 5 s and 4 s RNA cannot be present on the same molecules as rDNA, a few nucleotide sequences homologous to either 4 s or 5 s RNA could be interspersed throughout the high-density rDNA component. A single gene for 4 s or 5 s RNA for each 28 s and 18 s gene would yield hybridization levels with 3H-labeled 4 s or 5 s RNA of only 1 to 2% the level of that obtained with 3H-labeled rRNA. To test the possibility that some 4 s and 5 s genes might be present in rDNA fragments, large amounts of the rDNA satellite were purified for hybridization with 3H-labeled 5 s or 3H-labeled 4 s RNA in the presence or absence of their homologous cold RNA’s as well as non-radioactive rRNA. Only counts speci$cally competed out by the homologous cold RNA represent specific sequence homologies. The results are shown in Table 1. The 3H-labeled 5 s RNA preparation TABLE

Hybridization

of 3H-labeled 5 s RNA

1

and 3H-labeled 4 s RNA

with puri$ed rDNA

Cts/min hybridized [aH]RNA

0.2

pg

5 9

Unlabeled

RNA rDNAt

5 s DNA$

+ 20 pg 5 s

234 10 10

96 53 3

+ 20 pg 4 s

rDNAt 344 35 26

-

0.2 pg 5 9 0.2 pg 5 s

100 pg rRNA 100 pg rRNA

1.2pg4s 1.2pg4s 1.2 pg 4 s

100 pg rRNA 100 rg rRNA

4 s DNA1 107 70 11

t The high-densit,y fractions from about 2 mg of DNA centrifuged in a fixed-angle rotor were pooled and centrifuged a second time. This gradient was collected into 9 fractions. Each fraction was divided into seven equal parts and each portion was immobilized on an individual filter. Six of the filter sets were hybridized in 2 ml. of 4 x SSC containing the RNA’s listed above. The other set was hybridized with 1.8 pg of [3H]rRNA. More than 80% of rRNA hybridization occurred with one of the nine DNA filters (8000 cts/min); therefore, only counts hybridizing with this fraction have been scored. f The first CsCl fractionation enriched the rDNA complement about five- and tenfold over the 4 s DNA and 5 s DNA, respectively. However, enough 4 s and 5 s DNA was present to serve as controls for the competition experiments. Only counts hybridizing with DNA at the buoyant densities characteristic of 5 s and 4 s DNA were scored. The background radioactivity has been subtracted in each experiment.

bound 234 ats/min with the high density rDNA component (compared to 8000 cts/min by the 3H-labeled rRNA preparation). All but 10 of these counts were competed out by a 500-fold excess of cold rRNA; the remaining 10 counts were not competed out by the addition of cold 5 s RNA. In the same experiment the enormous excess of cold rRNA reduced specific 5 s RNA hybridization by about 40%, whereas the addition of cold 5 s RNA competed out more than 90% of these counts. In the case of 3H-labeled 4 s RNA, the homologous unlabeled RNA reduced the cts/min hybridized from 35 to 26 or about 0.1% of the level of 28 s and 18 s RNA hybridization (8000 cts/min). This extent of homology would be equivalent to less than one 4 s gene for every 10 copies of both 28 s and 18 s genes in the high-density rDNA component and is the limit of sensitivity of the method.

672

D. D. BROWN

AND

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(e) The presence of DNA homologous to 5 B RNA i,n 0-nu DNA Although 0-nu embryos do not synthesize detectable amounts of 5 s RNA (Brown, 1967), their DNA has normal complementarity to 5 s RNA (Fig. 8 and see also Fig. 13). This further demonstrates that 5 s DNA is not intermingled with rDNA in the genome of X. Levis.

Wild-type

DNA

I 3 0.4z 6 02d 0

IO Tube no.

FIG. 8. Hybridization of 3H-labelad 5 s RNA with wild-type and 0-nu DNA. About 25 pg of erythrocyte and 0-nu mutant DNA were denatured and centrifuged at neutral pH in a fixed angle rotor. The DNA filters from each fraction were hybridized with a mixture of wa8 0.17 pg of 3H-labeled 5 s RNA and 0.02 /.~g of [3H]rRNA in 5 ml. of 4 x SSC. Background optical -@-a--, Radioactivity (cts/min); -, 15 ots/min and has not been subtracted. density at 260 rnp.

(f) Saturation. levels of DNA hybridized with TRNA by the CsCl method The importance of prefractionation of DNA can be demonstrated by the following saturation experiments. The 3H-labeled rRNA of high specific activity was diluted with cold egg rRNA to a final specific activity of 25,000 cts/m.in/pg. DNA filters were prepared from six separate CsCl fractionations of varying amounts of DNA, and all the filters were hybridized together in the same vial with saturating amounts of 3H-labeled rRNA. The profiles of three of the six gradients are shown in Figure 9. Hybridization with the high-density DNA and with the bulk DNA were scored separately and plotted against the total amount of DNA in each fractionation in Figure 10. Although hybridization in the high-density region is directly proportional to the total DNA concentration, this is clearly not true of radioactive material bound by the bulk DNA. At lower DNA concentrations, the counts associated with the bulk DNA are greater relative to those associated with the high-density DNA. The relatively large contribution of this non-ribosomal radioactive material, which binds to small amounts of DNA, invalidates saturation curves using utiactionated DNA. A saturation experiment has also been performed on CsCl-fractionated DNA by increasing the amount of RNA while maintaining the DNA concentration constant. Again the need for prefractionation of DNA is apparent, since the relative extent of hybridization of radioactive RNA with bulk DNA and with satellite DNA is different at each RNA concentration (Fig. 11). The saturation value of O-O54o/oof the genome agrees with the saturation value of the previous experiment (0.057%) in which filters containing different amounts of DNA were hybridized with saturating amounts of RNA (Fig. 10).

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O-

O-

o-

0

4

”

Tube no. FIG. 9. Hybridization of saturating amounts of [3H]rRNA as a function of DNA concentration. CsCl fractionation in the fixed angle rotor was performed on 6 different amounts of native DNA; 5, 10, 20, 30, 40 and 100 pg. The gradients were fractionated into 9 fractions and each fraction was alkali denatured, neutralized and its DNA immobilized on a separate filter. All filters were hybridized with [sH]rRNA, the specific activity of which had been reduced from 400,000 ots/min/pg to 25,000 cts/min/pg by the addition of unlabeled rRNA. All filters were hybridized together in 5 ml. of 4 x SSC containing 5 pg/ml. of the t3H]rRNA, and three of the six hybridization patterns are plotted here: 10 clg DNA, -O-O-; 30pgDNA,--u--m--, and lOOpgDNA,--O-O-. The arrow refers to the peak tube of optical density. Background has not been subtracted (60 cts/min).

-6

-4

- 21

I

1

1

1

20

40

60

00

I

100

d0

/.q DNA FIG. 10. Hybridization of saturating amounts of [aH]rRNA with satellite and bulk DNA a8 a function of DNA concentration. The radioactivity hybridized in the satellite region (-•-•--) and bound under the DNA (- - O--O-) was scored from the experiment described in the legend of Fig. 9. Blank radioactivity was subtracted and both sets of values plotted against the total DNA which was fractionated in CsCl.

674

D. D. BROWN

(a)

0,039 *Id

AND

C. S. WEBER

b) 0.044%.

100

l!L d)

3 ,i 6

0.054%

0~050% 0.6-

!OO

I 2. 5 100 !OO 1 1

Tube no. Fm. 11. Hybridization of CsCl-fractionated DNA with increasing concentrations of [3H]rRNA. Four separate tubes each containing 50 pg of native DNA were fractionated in CsCl in a fIxed angle rotor; 9 fractions were collected from each tube. Each set of DNA filters was hybridized with a different concentration of [3H]rRNA (25,000 cts/min/pg). The percentage values refer to the calculated percentage of genome which hybridized with [3H]rRNA. This calculation was made by summing the count,s bound to high-density DNA and converting them to mpg RNA. The fraction of the genome is the amount of bound RNA divided by the total amount of DNA used for the fractionation. The amount of DNA was obtained by hydrolyzing the DNA from tho flters after they had been counted as described in the Materials and Methods section. The RNA concentrations were (a) 0.5, (b) 1.0, (c) 3.0 and (d) 5.0 pg/ml. -@-a--, Radioactivity (cts/min); optical density at 260 mp. -,

The difference in the amounts of [3H]RNA bound to the bulk DNA compared with the satellite is partly explained by the difference in specific activities of rRNA and DNA-like RNA after the dilution of the preparation with cold egg rRNA. Only the specific activity of rRNA is reduced by the added cold rRNA; any [3H]DNA-like RNA contaminant remains at the original specific activity. (g) Amount

of [3H]RNA

hybridization

relative to DNA

concentration

The amount of 3H-labeled rRNA, - 4 s RNA and - 5 s RNA which is hybridized is directly proportional to the amount of CsCl-fractionated DNA immobilized on filters even at subsaturating concentrations of the RNA. Sheared X. laevis DNA was centrifuged to equilibrium in an angle head rotor and nine fractions collected. Three samples of different volumes were taken from each of the nine fractions and the DNA was denatured and entrapped on separate Millipore filters as described. Each filter was cut in half and one set of half-filters was hybridized with [3H]rRNA, the other with 3JII-labeled 4 s RNA (Fig. 12, upper). The results plotted in the

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4s RNA

rRNA

I %

0.8 s S-4 d 6 0.4

0

IS

30

45

rug DNA FIQ. 12. Hybridization of subsaturating amounts of 3H-labeIed 4 s RNA and [3H]rRNA4 a8 a function of DNA concentration. About 70 pg of native, sheared DNA was centrifuged in C&l in a fixed angle rotor and fractionated into 9 fractions. Three sots of samples were taken from each tube equivalent to an initial input of 5.9 pg DNA, -@-a--, 15 pg DNA, --O--C-and 44pg DNA, -n---c]--, respectively. The DNA in each sample was immobilized on filters and the filters were split; one set of half-filters was hybridized with 1.8 rg [3H]rRNA in 4 ml. of 4 x SSC (upper left), the other set of half-filters was hybridized with 1.2 pg 3H-labeled 4 s RNA in 4 ml. of 4 x SSC (upper right). These RNA concentrations fill about 30% of the available DNA sites. In the lower Figure are scored the cts/min in tubes 5 and 6 from the [3H]rRNA hybridization (-e-a-), and the sum of cts/min in tubes 7, 8 and 9 from the 311-labeled 4 s RNA hybridization (-O-O-).

lower part of Figure 12 only scored the counts in the satellite region for rRNA hybridization and those under the DNA and slightly to the heavy side for 4 s RNA hybridization. The amount of RNA hybridized is directly proportional to the total amount of DNA immobilized on a set of filters. Furthermore, the linear response occurs even though the concentration of RNA in the reaction mixture does not saturate the homologous DNA sites. In this experiment only about 30% of the homologous DNA sites were hybridized. With respect to the rRNA hybridization, this direct relationship is maintained with CsCl-fractionated DNA ranging from 5 to 100 pg and with subsaturating amounts of [3H]rRNA which occupy as few as 2074 of the homologous DNA sites.

D. D. BROWN

676

AND

C. S. WEBER

(h) Relative abundance of rDNA and 5~ DNA in diflerent tissues Ritossa, Atwood, Lindsley & Spiegelman (1966) have shown that different somatic tissues of the chick have about the same proportion of their genome homologous to rRNA. DNA preparations from 1-nu embryos and from three wildtype sources which synthesize rRNA at different rates were compared for their relative abundance of rDNA and 5 s DNA by the CsCl-fractionation method (Fig.13).

rRNA A 0.4 L

2

Erythrocyte

/

OtJ

8 r-4

Early

6 (II

I

I \Liver

-

_.I.

\I

8 Heterozygote

gastrula

0

(l-flu)

:: Y .G .$ :: .O_ -u 2

02-

- 1000 0

0

IO

0 Tube

FIQ. 13. Relative About 30 pg of heterozygote (I-M) a mixture of 1.8 pg are plotted without density at 260 mp.

IO

no.

abundance of 5 s DNA and rDNA in the DNA from different tissues of X. Zaeuis. denatured DNA from X. Levis erythrocytes, liver, gastrula embryos and embryos were fractionated in CsCl in a Gxed angle rotor and hybridized with of 13H]rRNA and 0.2 pg of 3H-labeled 6s RNA in 8 ml. of 4 x SSC. The results background correction; -@-a-, radioactivity (cts/min); -, optical

The proportion of rDNA as well as 5 s DNA in the wild-type tissues are not detectably different, even though these tissues vary enormously in their ability to support rRNA synthesis. The 1-nu DNA has half the amount of rDNA but a normal complement of 5 s DNA. In marked contrast to somatic tissues, oocytes have many extra copies of rDNA relative to the bulk of nuclear DNA (Brown $ Dawid, 1968).

4. Discussion (a) Notes on the techniques (i) RNA purij2ation Two steps in [3H]rRNA purification lowered the background of hybridization as monitored by binding to blank tllters, and the levels of radioactivity which was bound to the bulk DNA. These steps were the MAK purification and the final filtration of the [3H]rRNA through a Millipore filter before its use for hybridization. Filtration

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of the [3H]rRNA reduces the non-specific binding to all filters including blank filtera. Without the MAK fractionation step the C3H]rRNA preparations hybridized extensively with the bulk DNA, presumably due to DNA-like RNA contamination. Whether the residual binding of [3H]rRNA preparations with the bulk DNA was due to some remaining DNA-like RNA in the preparation or to some other contaminant is not known. The [3H]rRNA preparations bind to some extent with the bulk DNA from even the most heterologous sources including bacterial DNA’s of widely different base composition as well as phage DNA’s. About one-third as much radioactive material binds to these heterologous DNA’s compared to that which binds to the bulk of X. laevti DNA. None of this “hybridization” is reduced by the addition of excess cold rRNA. (ii) DNA pur@cation In order to use the method of Gillespie & Spiegelman (1965) with confidence, each preparation of DNA must be carefully assayed, not only for the proportion of DNA which is immobilized by the nitrocellulose filter, but also for the proportion of DNA which is retained after hybridization. In our experience, unde6ned impurities interfere with immobilization of DNA and cause variable results. Fractionation of DNA in CsCl further purified DNA preparations and yields remarkably uniform results in the subsequent immobilization and hybridization steps. For example, RNase present in the original unfractionated DNA preparation is removed completely by the centrifugation-filtration method. (iii) Specijkity and sen,sitizGty of the method The specificity of the method resides in using CsCl fractionation of DNA to separate DNA fragments of different buoyant densities. The CsCl fractionation, coupled with the use of homologous cold RNA as a competitor, can distinguish between specific hybridization and ‘Lnon-specihc” binding which is not reduced by the unlabeled RNA competitor (Figs 4 to 7). The importance of CsCl fractionation is evident in the saturation experiments, where non-homologous binding can be scored separately (Fig8 9 and 11). Since the non-homologous binding is not linearly related to DNA (Fig. lo), large errors in percentage genome at saturation, particularly at low DNA concentrations, will be made unless the specific counts alone are scored. The high specificity also increase8 the sensitivity of the method. If O-W DNA retained one of the four hundred and fifty 28 s and 18 s sequences found in the wild-type haploid genome, it would have bound 4 cts/min of [3H]RNA in the experiment recorded in Figure 4. As many as three remaining genes would have been detected in this experiment. Therefore, more than 99% of the DNA homologous to 28 s and 18 s RNA has been deleted by this single mutation. The experiment demonstrates that essentially all of the rDNA is clustered and band8 as a highdensity satellite in CsCl. Another important conclusion made possible by the CsCl method is the demonstration that less than 0.1% of the nucleotide sequences of the high-density rDNA satellite are homologous with either 5 s RNA or 4 s RNA (Table 1). The level of sensitivity here is such that there cannot be more than one DNA sequence homologous to 4 s or 5 s RNA for each 10 sequences homologous to 28 s and 18 s RNA in this high buoyant density DNA component.

678

(iv) Determination

D. D. BROWN

of the proportion

AND

of a DNA

C. S. WEBER

preparation

homologous to rRNA

The range of amounts of total DNA that can be included in a centrifugation in limited only by the decreased effectiveness of satellite separation discussed before (Fig. 1). Large numbers of filters do not impede the hybridization reaction. The extent of specific hybridization of rRNA (Figs 1, 12 and 13) and 4 s RNA (Fig. 12) is directly proportional to the amount of homologous DNA on filters even at subsaturating RNA concentrations. Similar results have been obtained for hybridization of 5 s RNA, as can be seen in Figure 13. As a result of this relationship, an unknown DNA preparation can be assayed for the amount of homologous DNA as long as a set of filters containing a known amount of CsCl-fractionated DNA (in which the percentage genome homologous to the specific RNA is known) is included in the same hybridization reaction. The linear nature of this response has been used to quantitate preparations of DNA greatly enriched for rDNA, such as oocyte DNA, which has hundreds of extra copies (Brown & Dawid, 1968). (b) Clustering of 28 s and 18 8 genes The clustering of genes for 28 s and 18 s RNA may be a widespread phenomenon. The first rRNA-DNA hybridization studies by Yankofsky & Spiegelman (1962) led them to conclude that the several genes for rRNA in E. coli are clustered. A similar conclusion was reached for Drosophila melanogaster by Ritossa & Spiegelman (1965), who showed that the amount of rDNA Ifer cell is proportional to the number of nucleolar organizers. They estimated that about 65 of the 28 s and 18 s genes were clustered at each nucleolar organizer. Furthermore, “bobbed” mutants of D. melanogaster, the defect of which is known to map at the nucleolar organizer locus on the X chromosome, were found to contain partial deletions of rDNA (Ritossa et al., 1966). Birnstiel et al. (1966) reported that 0.04 to 0.07a/0 and 0.025 to O+O4o/oof X. laevis DNA is homologous with 28 s and 18 s RNA, respectively. Since an 18 s gene should be half the size of a 28 s gene, these values suggest that there are about equal numbers of these two genes in a redundant cluster of rDNA. In these studies, about 0.057% of the DNA of X. taevis was found to be homologous with a combination of 3H-labeled 28 s and 3H-labeled 18 s rRNA. From the known amount of DNA per haploid genome (3 t+g, Dawid, 1965) and the estimated molecular weights of 28 s RNA and 18 s RNA (1.4 and 0.7 x lo6 daltons, respectively; U. Loening, personal communication), there are about 450 of the 28 s and 18 s genes in each haploid complement. Essentially all of these redundant sequences are clustered together on part of a single autosome, since they are deleted together by the anucleolate mutation. The accompanying paper demonstrates that the 28 s and 18 s genes are highly intermingled within this redundant cluster and probably alternating (Brown & Weber, 1968). (c) The 5 s genome

DNA homologous to 5 s RNA clearly is not intermingled among the multiple redundant 28 s and 18 s RNA genes. This conclusion has been reached from three separate considerations. First, although 0-nu embryos are depleted of more than 99% of their rDNA, they have a normal complement of 5 s DNA (Fig. 8). Second, oocytes greatly enriched in DNA homologous to 28 s and 18 s DNA are not enriched for 5 s DNA

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(Brown & Dawid, 1968). Third, DNA homologous to 5 s RNA has a different buoyant density than rDNA in CsCl (Fig. 2). Although approximately equal numbers of nucleotide sequences for 28 s and 18 s RNA are present (Birnstiel et al., 1966), the extent of DNA homologous to 5 s RNA in X. Levis can accommodate an enormous number of genes. Although saturation experiments have not been performed, at least 0.05% of the DNA of X. laevis is homologous to 5 s RNA. If its molecular weight is approximately 35,000 daltons (Rosset, Monier & Julien, 1964), there must be at least 27,000 genes in each haploid genome . Several features about the DNA homologous to 5 s RNA remain unexplained. Although 5 s RNA has a high guanylic-plus-cytidylic acid content, its homologous DNA bands on the light side of the bulk DNA when either native DNA or denatured DNA is centrifuged at neutral or alkaline pH in CsCl (Fig. 2). The 5 s DNA bands on the light side when the DNA molecules range in molecular weight from O-5 to 50 x lo6 daltons. The buoyant density of this DNA is the same when obtained from different tissues of X. luevis (Fig. 13). Its density could be shifted to higher values by only one technique to date, namely by hybridization with 5 s RNA before centrifugation (Brown & Weber, unpublished observations). This experiment indicates that the multiple 5 s genes are clustered to some extent, a fact which makes their low buoyant density even more puzzling. One hypothesis is that the 5 s genes are interspersed with relatively short stretches of DNA of very low buoyant density. The synthesis of 28 s and 18 s RNA is co-ordinated by transcribing them together as part of a single polycistronic precursor molecule (Brown & Weber, 1968). However, 5 s RNA cannot be derived from these same precursor molecules since its homologous DNA is not intermingled with 28 s and 18 s DNA. Recent findings of a pool of 5 s RNA in HeLa nuclei (Knight & Darnell, 1967) support its independent synthesis from 28 s and 18 s RNA. The accumulation of 5 s RNA appears to be controlled by the rate of accumulation of 28 s and 18 s RNA in both oogenesis and embryogenesis of X. Eaevis. Oocytes synthesize 28 s and 18 s RNA at very high rates using extra replicas of their genes as templates; the 5 s RNA accumulates co-ordinately with 28 s and 18 s RNA in oocytes even though its genes are not amplified (Brown & Dawid, 1968). AnucIeolate embryos without 28 s and 18 s genes do not accumulate 5 s RNA even though the DNA homologous to 5 s RNA is present. (d) The 4 s genome Saturation experiments show that about 0.016% of the DNA of X. laevis is homologous to 4 s RNA (Brown & Weber, unpublished results); this represents about 1000 genes for 4 s RNA in the haploid genome. The base composition of 4 s RNA is 60% guanylic-plus-cytidylic acid (Brown & Littna, 1964), so that if these multiple genes were clustered the 4 s DNA should band as a high-density satellite like rDNA. Since the DNA homologous to 4 s RNA bands coincidently with and to the heavy side of the bulk DNA (Figs 3 and 12), we conclude that a portion of the 4 s DNA is clustered and the remainder is dispersed throughout the genome. There is less than one 4 s gene for every ten copies of the 28 s and 18 s genes in the rDNA satellite. Competition experiments indicate that there are no homologous sequences shared between 4 s and rRNA (Fig. 6) or between 4 s and 5 s RNA (Fig. 7). These studies agree with those of Ritossa et al. (1966) who showed that the genes for tRNA in

680

D. D. BROWN

AND

D. melanogaster do not map at the nucleolar throughout its genome.

C. S. WEBER organizer

sites and may be dispersed

REFERENCES & Thomas, C. A., Jr. (1965). J. Mol. BioZ. 11, 476. L. (1967). In Cell D$erentiation, ed. by A. V. S. DeReuck & J. Knight, p. 178. J. & A. Churchill Ltd. L., Wallace, H., Sirlin, J. & Fischberg, M. (1966). Nut. Cancer Inst. Monog.

Berms, K. I. Birnstiel, M. London: Birnstiel, M. 23, 431. Brown, D. D. (1965). In Developmental and Metabolic Control Jle~ka~~ierns and NeopZa&a, p. 219. Baltimore: Williams BEWilkins Co. Brown, D. D. (1967). In Current Topics in Developmental Biology, ed. by A. Monroy & A. Moscona, vol. 2, p. 47, New York: Academic Press. Brown, D. D. & Dawid, I. B. (1968). Science, 160, 272, Brown, D. D. & Littna, E. (1964). J. Mol. BioZ. 8, 669. Brown, D. D. & Littna, E. (1966). J. Mol. BioZ. 20, 95. Brown, D. D. & Weber, C. S. (1968). J. Mol. BioZ. 34, 681. Dawid, I. B. (1965). J. Mol. BioZ. 12, 581. Elsdale, T. R., Fischberg, M. & Smith, S. (1958). Exp. Cell h!e8. 14, 642. Flamm, W. G., Bond, E. & Burr, H. E. (1966). Biochim. biophys. Actu, 129, 310. Galibert, F., Larsen, C. J., Lelong, J. C. & Boiron, M. (1965). Nature, 207, 1039. Gillespie, D. & Spiegelman, S. (1965). J. Mol. BioZ. 12, 829. Knight, E., Jr. & Darnell, J. E. (1967). J. MOE. BioZ. 28, 491. Mandell, J. D. & Hershey, A. D. (1960). Analyt. Biochem. 1, 66. Marmur, J. (1961). J. Mol. BioZ. 3, 208. Nieuwkoop, P. D. & Faber, J. (1956). Normal Tableo of Xenopus laevis (Da&in). Amsterdam: North Holland Publ. Co. Ritossa, F. M., Atwood, K. C., Lindsley, D. L. & Spiegehnan, S. (1966). Nat. Cancer In-d. Monog. 23, 449. Ritossa, F. M. & Spiegelman, S. (1966). Proc. Nat. Acad. Sci., Wash. 53, 737. Rosset, R., Monier, R. & Julien, J. (1964). Bull. Sot. China. BioZ. 46, 87. Wallace, H. & Birnstiel, M. L. (1966). Biochim. biophys. Acta, 114, 296. Yankofsky, S. A. & Spiegelman, S. (1962). Proc. Nat. Acad. Sci., Wash. 48, 1466.