Preferential transcription of Xenopus laevis ribosomal RNA in interspecies hybrids between Xenopus laevis and Xenopus mulleri

J. Alol. Biol. (1973) 80, 217-228

Preferential Transcription of Xenopus laevis Ribosomal RNA in Interspecies Hybrids between Xenopus laevis and Xenopus mulleri TASUKU HONJO~ AND RONALD H.REEDER Department of Embryology Carnegie Institution of Washington 115 W. University Parkway Baltimore, Md 21210, U.X.A. (Received 19 March 1973) Rihosomal RNA synthesized by hybrid frogs from the cross between Xenopus Zaevis and Xenopzce mull& was analyzed by molecular hybridization with purified ribosomal DNA from each species. Although the 18 S and 28 S rRNA sequences are indistinguishable between these two species, the remaining 10% of the 40 S rRNA precursor molecule of each species hybridizes about tenfold more efficiently to homologous rDNA than to heterologous rDNA$. Using an assay based upon this fact, we show that in hybrid frogs X. Zaevis rDNA is transcribed preferentially and X. mulleri rDNA is repressed. X. mulleri rDNA is repressed regardless of which species is the female parent. The repression is nearly complete throughout early embryogenesis until the swimming tadpole stage, after which a low level of X. mulleri rRNA synthesis is detectable in the total embryo population. Some adult frogs made no detectable X. mulleri rRNA, whereas others were found that synthesized substantial amounts. ‘Transcription of X. m&e& rDNA is repressed in embryos from the cross of a X. Zaevis female, heterozygous for the rDNA deletion mutation, and a wild type X. mulleti male. Half of these embryos contain only X. mulleri rDNA. The X. mulleri rDNA is transcribed eventually in these embryos but the onset of rR,NA synthesis is much later than in wild type X. mulleri embryos. In the reverse cross (female X. mulleri x male X. Zaevis heterozygote) turn-on of X. mulleri rR,NA synthesis was not delayed. The results of these four types of crosses indicate that either X. Zaevis rDNA or X. Zaevk maternal cytoplasm can each repress expression of X. mulleri rDNA in hybrid embryos. In the presence of X. laevk rDNA the repression can be permanent. The repression by X. Zaevis cytoplasm is transient and usually reversible.

1. Introduction Wild type frogs of the species Xenopw laevis and Xenopus mulleri contain two prominent nucleoli per cell throughout most of embryogenesis. Bladder & Gecking (1972), and Cassidy & Blackler (unpublished results) have recently shown, however, that T Present address: Laboratory of Molecular Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Md 20014, U.S.A. $ Abbreviations used: rRNA, 18 S and 28 S ribosomal RNA; rDNA, the DNA containing the zeclnenoes for rRNA

as well as spacer sequences (Dawid 217

et nl., 1970).

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F, hybrids between these two species have only one nucleolus in the large majority of their cells, suggesting that the nucleolar organizer of one of the two species is somehow inactivated by the presence of the other. Similar observations on nucleolar dominance have been made in interspecies hybrids within the plant genera Crepis, Ribes and Xalix (Navashin, 1934; Wilkinson, 1944; Keep, 1962; Wallace & Langridge, 1971). Since it is well established that the nucleolus is the site of rRNAt synthesis in interphase cells (for a review see Birnstiel et al., 1971), the inference has been made that the rDNA of one species is repressed in the hybrid cells. If this inference is correct, such cells may provide a useful system in which to analyze the control of rRNA synthesis in eukaryotes. The present paper presents a biochemical description of nucleolar dominance in hybrids between X. Levis and X. mulleri. These hybrids have an obvious advantage for this type of study, since the rDNA from each species has been isolated in pure form and their structures have been studied extensively (Dawid et al., 1970; Brown et al., 1972; Birnstiel et al., 1971). Both the rDNA and the rRNA precursors of the two species can be distinguished (Brown et al., 1972). In X. laevis-X. mulleri hybrid animals X. laevis rRNA is synthesized almost exclusively, even though the rDNAs from both species are present. We also provide evidence that turn-on of ribosomal genes occurs at a specific developmental stage during early embryogenesis of hybrid embryos. A preliminary report of this paper has been published (Honjo & Reeder, 1973).

2. Materials and Methods (a) Production of hybrid animals Procedures for producing and maintaining hybrids between laevis$ and mu&& have been described by Blackler & Gecking (1972). Some of the hybrids used in this study were generously donated by Dr A. W. Blackler and some were gifts of Dr D. D. Brown. In desoribing the crosses and genotypes we follow the convention of giving the species of the female first. The diploid genotypes of the rRNA of wild type 1asvLs and mu&& are expressed by E/I and m/m, respectively. For example, l/l- indicates laevb heterozygous for the nucleolar mutation that deletes the rDNA (Elsdale et al., 1958; Wallace & Birnstiel, 1966). Embryos were staged according to the Tables of Nieuwkoop & Faber (1956). (b) Synthesis and hybridization of radioactive RNA Isolation of nuclei, synthesis of nuclear RNA ilz vitro, and the isolation of RNA for molecular hybridization have been described previously (Reeder & Roeder, 1972). In the present work the methods were slightly modified. The initial homogenization of either embryos or adult tissue was carried out in 0.25 M-SUCroSe containing 0.1 M-KCl, 0.01 M-Tris*HCl (pH S), and 5 mM-MgCls. Triton Xl00 (O-lo/)o was included for adult tissues. Reaction mixtures for synthesis of nuclear RNA contained 0.4 M-KC1 and 2 pg a-amanitin/ ml in addition to the other ingredients and they were incubated for 40 min. RNA was hybridized to DNA immobilized on filters at 70°C overnight, as described by Brown & Weber (1968), in 0.6 M-N&I, 0.2 M-Tris.HCl (pH 8) and 4 mM-EDTA. (c) Iso.lu&n of nucleic mids and mea-suremeti of TDNA Purified rDNA isolated from young ovaries of either laevk or mull.& was the gift of Dr D. D. Brown (Brown et al., 1972). Unlabeled 18 S and 28 S rRNA was isolated as described previously (Brown L%Weber, 1968). Bulk somatic DNA was isolated as described

t See footnote on p. 217. $ For the sake of brevity of this paper,

the two species will be referred

to as Zae& and mJ?eri

for the rest

rRNA

SYNTHESIS

IN HYBRID

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219

previously (Brown & Weber, 1968) from blood obtained by clipping a toe from a single animal. For embryos, a small number were removed from the population and total DNA was extracted from them. The analytical procedure for measuring Lewis and muEZeri rDNA in bulk DNA from hybrid frogs is based on the fact that the nucleotide sequences of the rDNA spacer regions differ between the 2 species. The method has been described by Brown BEBlackler (1972) and requires as little as 10 pg of bulk DNA for each analysis. Measurements on adults were done by Dr D. D. Brown. (d) Other materials Tissue culture cells of laevk were grown as described previously (Brown & Weber, 1968). [3H]CTP (20 Ci/mmol) was purchased from Schwarz/Mann, and E-[~~P]CTP (17 to 35 Ci/mmol) from International Chemical and Nuclear Corp. a-Amanitin was a gift from Dr R. G. Roeder.

3. Results (a) Presence of X. laevis and X. mulleri

rDNA

in the F, hybrids

We have found, in agreement with Brown $ Blackler (1972), that in crosses between twao wild type animals the rDNA composition of the hybrids is predictable from the genotypes of the parents, i.e. each nucleolar organizer behaves like a Mendelian factor. In crosses such as Levis (l/l-) x mulleri (m/m), where one parent carries the nucleolar deletion, the offspring usually fell into two classes as expected, those with equal amounts of laevis and mulleri rDNA (l/m) and those with only mulleri rDNA (l-/m). In occasional crosses of this type, however, the l-/m animals did not survive, leaving only those with the l/m genotype (see section (d), below). (11) Measurements

of the relative synthesis of X. mulleri in isolated nuclei

versus X. laevis rRNA

The RNAs of both species of Xenopus are initially transcribed as large precursor molecules with a molecular weight of about 2.5 x lo6 (Dawid et al., 1970). About 2.2 x 10s molecular weight of this molecule is accounted for by sequences for 18 S and 28 S rRNA which cannot be distinguished between laevis and mulleri. The remainder of the precursor molecule, however, differs considerably in sequence between the two species and hybridizes about tenfold more efficiently to homologous rDNA. than to heterologous rDNA (Brown et ul., 1972). To determine which species of rRNA precursor is being synthesized in a hybrid animal, authentic laevis 3Hlabeled precursor was mixed with 32P-labeled precursor from the hybrid animal and hybridized to purified laevis rDNA and to purified mulled rDNA, in the presence of excess unlabeled 18 S and 28 S rRNA. From the isotope ratios binding to the two types of rDNA, the ratio of Levis to mdleri rRNA in the hybrid animal can be determined. To label precursor rRNA molecules to a sufficiently high specific radioactivity, nuclei were isolated either from the liver of a single animal or from a group of embryos, and the endogenous RNA polymerase was allowed to elongate rRNA chains in vitro. As shown previously (Reeder $ Roeder, 1972), nuclei make rRNA for about 20 minutes under these conditions and then stop. The available evidence suggests that there is no reinitiation of rRNA chains. In the presence of a-amanitin about 90% of the total RNA synthesized is rRNA. This rRNA has been shown to be transcribed from the H-strand of the rDNA, to consist largely of 18 S and 28 S rRNA seque:nces, and to contain additional sequences that are not 18 S or 28 S. It thus

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contains nucleotide sequences similar to those found in authentic precursor rRNA molecules. Using rRNA labeled in isolated nuclei we constructed a standard curve relating the isotope ratios bound by the two rDNAs to the proportion of Eaevis and mulleri rRNA in a mixture. In separate reaction mixtures, nuclear RNAs from embryos of laevis and mulleri were labeled with 32P. The 32P-labeled RNAs were then mixed in several proportions. Nuclei from laevis tissue culture cells were used to synthesize 3H-labeled nuclear RNA as a standard. To each sample of 32P-labeled RNAs was added an equal amount of 3H-labeled standard RNA and 100 pg of unlabeled 18 S and 28 S rRNA. Each doubly labeled RNA mixture was hybridized to a filter containing mulleri rDNA and to a filter containing laevis rDNA in the same vial. The result of such an experiment is that for each artificial mixture of 32P-labeled m,ulleri and laevis RNA one obtains a 3H/32P ratio bound to laevis rDNA (L ratio), and a 3H/32P ratio bound to mulleri rDNA (M ratio). In the case where the [32P]RNB is lOOo/ laevis, the L ratio should be the same as the M ratio and, therefore, the ratio of the L ratio to the M ratio (L/Jfratio) shouldequal unity. At the other extreme, when the [32P]RNA is lOOo/o mulleri, the L ratio is greater than the M ratio. We have determined empirically that the L/M ratio is close to 158 at this extreme. A standard curve obtained by the above method is shown in Figure I. The line drawn through the experimental points in Figure 1 is a theoretical curve, the derivation of which is described in the Appendix. The effect on the observed L/M ratio of varying the amount of input RNA or the amount of rDNA on the filters is shown in Figure 2. In these experiments the amount of input RNA was varied over a 55-fold range without significantly affecting the outcome of the measurement (Fig. 2(a) and (b)). Varying the amount of laevis rDNA

1. Standard curve for the measurement of mulleti rRNA synthesis in hybrid frogs. Construction of the curve is described in the text. The points am from individual measurements on artificial mixtures of kzevk and mzclleri nuclear RNA. Each symbol represents s separate experiFIG.

ment. The line ia from e theoretical

caloulation

described in the Appendix.

rRNA

0 .---I-L. 0 2

4

SYNTHESIS

LL.6 8

[32~1~~~

IO Input

IN

0

20

(cts/mln

HYBRID

40

60

x IO-~)

FROGS

I I

0 /mm

221

-L--12

3

rDNA/fllter(~g)

FIQ. 2. Effect of varying RNA and DNA amounts on meesurement of mulleri rRNA. Each point is the result of r~ separate hybridization reaction containing unlabeled 18 S and 28 S rRNA (100 pg), 3H-lebeled Zaewis nuclear RNA (191,000 cts/min) and 3aP-lmbeled nuclear RNA which was partly from Zaevis and partly from mulleti as specified. Each reaction also contained a filter with 2 pg of mulleri rDNA, a filter with a specified amount of Zaewis rDNA, and a blank filter (no DNA). Percentage mulleti rRNA in the sap-labeled nuclear RNA was calculated from the isotope ratios bound to the 2 rDNA filters by use of the standard curve shown in Fig. 1. The broken lines show the percentage of muZZeri nuclear RNA in the input [3aP]RNA. (a) Effect of varying sap-labeled nuclear RNA input, Zaevia rDNA constant at 1.1 pg/fllter. The input [32P]RNA ww 46% from mulleti nuclei in each case. (b) Same ae (a) except that input [32P]RNA was 21 y0 from mulleri in each case. (c) Effect of varying the amount of Zaevis rDNA/ filter, input [3aP]RNA held constant at 93,800 cts/min. The input [3aP]RNA was 46% from mulleri in each case.

per filter over a sixfold range also had no significant effect (Fig. 2(c)). The average error of all the determinations shown in Figure 2 was 5.7%. The sensitivity of the method was checked by making measurements on different bztches of pure kzevis and pure mulleri nuclei. From the measured L/M ratio and the use of Figure 1 we calculated the apparent percentage of mulleri rRNA synthesized in each batch. The values are listed in Table 1. The values range from 0 to 3 TABLE 1 rRNA synthesized by wild type laevis and mulleri Animals -laevisl Zaewis, laevia3 muZZer& mu&Wi, muZZe+ 1: 1 mixture (stage 16-17)t 1: 1 mixture (stage 37-38)t

Input RNA (cts/min) C3H]RNA [3aP]RNA

Hybridized to Zueti rDNA 3H sap

RNA

(cts/min) to muZZeri rDNA sH s=P

mulleri rRNA (%)

200,000 191,000 191,000 200,000 191,000 382,000

162,000 260,000 341,000 163,600 96,000 287,000

498 6360 7690 670 9170 26,100

236 2460 2330 32 68 134

31 2380 1400 47 1260 3380

21 907 427 273 1180 1970

3 0 0 96 100 97

191,000

66,800

6900

78

881

175

55

191,000

79,000

2490

170

1189

609

36

[aaP]RNA was synthesized by nuclei isolated from whole embryos except that adult liver W&S employed in Zaeuis, and mdleri,. 100 pg of 18 S and 28 S Zuewis rRNA and 3H-labeled standard RNA synthesized by la&a nuclei were added to each [3aP]RNA preparation. The mixture ww hybridized to nitrocellulose filters containing approximately 1 pg of each Zaevis and muZZeri rDNA. t Equal numbers of Zaeti and mulleti wild type embryos were mixed and their nuclei isolated and analyzed together for rRNA synthetic capwity.

222

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REEDER

for pure laevis and 96 to 100 for pure mulleri nuclei. The lower limit of detection is therefore about 5% mulleri rRNA in the presence of 95% luevis rRNA. The e6iciency of hybridization of the standard laevis RNA varied from one batch of RNA to another (see Table 1). This variation was most likely due to the fact that both RNA synthetic oapacity and content of unlabeled precursor rRNA varied from one batch of nuclei to another, resulting in differing RNA specific radioactivities. (c) rRNA synthesis in F, hybrids from the cross X. laevis (l/l) X X. mulleri (m/m) Measurements on RNA synthesized in nuclei from embryos of this cross are shown in Table 2. Prom the earliest stage tested, stage 15, and thereafter until about stage 43, the mulleri rDNA was repressed completely. At stage 43 and beyond there was detectable mulleri rRNA synthesis in the population of hybrid embryos. In contrast, wild type mulleri embryos at similar stages make rRNA as actively as do wild type Levis embryos. This is shown by an experiment in which equal numbers of stage 15 to 17 luevis and mulleri embryos were mixed, their nuclei isolated and found to synthesize 550/, of their total rRNA as mulleri rRNA (Table 1). A similar experiment, in which equal numbers of stage 37 to 38 embryos were mixed, showed 36O/” mulleri rRNA synthesis. We also measuredrRNAsynthesisinadulttissues from several hybrid frogs (Table 3). In liver nuclei from three of the animals, mulleri rRNA synthesis was either absent or very low. In the other four animals between 17 and 50% of the rRNA synthesized was mulleri. (d) rRNA synthesis in F, hybrids from the cross X. laevis (Z/l-) X X. mulleri (m/m) This type of mating results in an embryonic population with a one to one mixture of the two genotypes, l/m and 1-/m. Since the two genotypes cannot be distinguished morphologically, mixed populations of embryos were analyzed for rRNA synthesis. It was assumed that any mulleri rRNA synthesis would be derived from the l-/m genotype, since the l/m genotype makes no mulleri rRNA before stage 43 (Table 2).

TABLE 2 rRNA synthesized by laevis x mulleri hybrid embryos

Stage

Input [3aP]RNA (cts/min)

1‘5-17 28-30 37-39 43-46 48-49

632,000 178,000 216,000 280,000 104,000

Hybridized to Zaewti rDNA 3H 32P 20,100 9710 10,900 12,900 12,100

8600 4290 6330 13,000 1800

RNA

(cts/min) to mulleri 3H 1960 1420 1380 1630 1930

rDNA =P 728 686 916 3290 660

mulleri rRNA (%I 0 0 1 8 10

100 pg of 18 S and 28 S Zaewia rRNA and 3H-labeled standard RNA (191,000 cts/min) synthesized by Zaevis nuclei were added to [32P]RNA of each stage, except stage 16-17 where 382,000 cts/min of [3H]RNA were used. The RNA mixtures were hybridized to filters oontaining approximately 1 pg each of lawis and mu&& rDNA.

rRNA

SYNTHESIS

IN

HYBRID

223

FROGS

3

TABLE

rRNA synthesized by laevis x mulleri hybrid adults

Crosses

Hybridized to Zaetis rDNA 32P 3H

Tissue

Liver Liver Liver Liver Liver Liver Liver Kidney

RNA

(cts/min) to mulleri 3H

199 77 783 1455 192 733 2380 89

311 1270 4364 4650 4643 4398 3250 2100

mulleri

rDNA 32P 38 30 697 279 302 199 2140 259

96 319 1615 239 1288 213 407 495

rRNA (%) 0 4 8 17 27 28 34 49

A separate animal was used for each analysis except for cross no. 3, where both liver and kidney were taken from a single animal. Cross no. 1 was Z/Z- x m/m. Somatic DNA of the animals used from cross no. 1 was shown to have the genotype Z/m. Crosses no. 2 and no. 3 were Z/l x m/m. RN.4 from hybrid nuclei was labeled with 32P and standard Zaewia RNA was labeled with 3H.

50

40 2 6 2 (LL

30

1

20

8 IO

0

I

20

t

L

I

SO 60 40 Time after fertlluatlon jh)

40 IO 15 19 26 32 35 I-III_--\ f‘\ Hatchtng Gostrulo Neurula I Heart beat

42

I

100

120

444546 / Feedmg

N~euwkcop-Faber stages FIQ. 3. Synthesis of muJleri rRNA during early development in Xenopus hybrids. :Nuclei were isolated from embryos at various stages and analyzed for their ability to synthesize rRNA as described in the text. Embryos from the same mating have the same symbol. Open symbols, dotted line: embryos from the cross E/l- x m/m. Closed symbols, solid line: embryos from the cross Z/Z x m/m. Data for this cross were taken from Table 2.

T. HONJO

224

AND

R. H.

REEDER

The results are shown in Figure 3. At stage 13 to 14 there was almost no detectable mulleri rRNA synthesis. After stage 13 to 14, mulleri rRNA synthesis gradually increased relative to Levis until stage 43 when mulleri rRNA accounted for about 50% of the rRNA synthesized in the total embryo population. A portion of each batch from this cross was analyzed cytologically to see if there was any selective death of one genotype or the other during development. This analysis is based on the observation by Cassidy & Blackler (unpublished results) that at stage 15 to 20 the l-/m genotype has no nucleoli, whereas the l/m genotype has one per cell. For latter stages only batches of embryos with high survival rates (greater than 80%) were used. The data in Figure 3 are derived from six separate matings. A seventh was discarded because the embryos showed significant loss of the l-/m genotype before stage 19. (e) rRNA synthesis in hybrid& where X. mulleri is the female parent In both of the crosses described so far the luevis rDNA was donated by the female parent, which also donated the cytoplasm of the zygote. We therefore tested animals from the reverse*cross, mulleri (m/m) x Eaevis (l/l), to assess the influence of egg cytoplasm on rDNA expression. The results are shown in Table 4. As early as stage 15, embryos of the m/l genotype synthesized luevis rRNA exclusively. With the possible exception of stage 22 to 26, embryos of the m/l genotype repress mulleri rRNA synthesis as completely as do embryos of the reverse (l/m) genotype. Only one adult from this type of cross was available and both its liver and kidney showed complete repression of mulleri rRNA (Table 4). From these results we conclude that transcription of laevis rDNA is dominant regardless of which species is the female parent. In one experiment we also analyzed embryos from the cross mulleri (m/m) x luevis (l/l-). As early as stage 15 to 20, these embryos were making a significant amount TABLET rRNA synthesized by mulleri

CTOS53S

1 2 2 3 1 3 3 3 4 4 6

stage

16-19 16-20 22-26 29-32 36-38 39-41 44-46 48 One adult liver One adult kidney 16-20

Hybridized to Eaevis rDNA aH =P 13,700 3210 17,100 13,400 16,100 14,300 12,200 12,600 3280 2660 606

8640 106 21,700 6290 42,900 6330 6900 2340 3680 237 276

x laevis hybrids RNA

(ote/min) to mu&-i SH 2300 496 1240 1940 1790 1900 1200 2060 1030

rDNA s=P 1310 13 6160 1380 6660 1430 1190 779 1780 92 304

mu&A rRNA (%I

0 0 19 4 2 8 8 8 4 0 30

Crosses 1 to 4 were m/m X 111produced by arti&ial fertiliz&ion. Cross 6 wa8 m/m x 1/l- and was done using trenmniasion aGxn& (Blwkler & Geaking, 1972). RNA from hybrida WM labeled with S’P and standard Zaevis RNA with 8H.

rRNA

SYNTHESIS

IN HYBRID

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225

of mulleri rRNA (Table 4) in contrast to the reverse cross shown in Figure 3. The embryos from this cross are a mixture of two genotypes, m/l and m/E-. Since at the same stage (16 to 19) the m/l genotype embryos make no mulleri rRNA, it is clear that the m/l- embryos are engaged in active synthesis of mdleri rRNA. 4. Discussion (a) Repression

of X. mulleri rDNA

The results of the four types of matings can be summarized as follows: when the hybrid receives both Levis and mulleri rDNA (I/ m and m/l), transcription of the lae,vis rDNA is usually dominant and the mulleri rDNA is repressed. Repression of mulleri rDNA occurs as early as rRNA synthesis can be measured in embryos (stage 15). We have not been able to obtain enough nuclei from hybrid embryos before stage 15 to make reliable measurements. It appears likely, however, that mulleri rRNA synthesis was never turned on in the embryos. In most cases, the repression extends throughout embryogenesis and is found in adult tissues as well. When Levis rDNA is present, the paternal cytoplasm has little effect on the severity of the repression with the possible exception of stage 22 to 26 of the m/l hybrids (Table 4). Tho detection of significant mulleri rRNA synthesis in this one experiment is at present unexplained. When the hybrid receives only mulleri rDNA and Levis rDNA is deleted, two different results can occur. (1) If the zygote receives Levis ooplasm (hybrid genotype l-/m), mulleri rRNA synthesis is transiently repressed and then is gradually turned on. (2) If the zygote receives mulleri ooplasm (hybrid genotype m/l-), turn-on of mulleri rRNA synthesis is not delayed. These results suggest that mulleri rRNA synthesis can be repressed in two apparently different ways. One type of repression is due to the presence of kzevis rDNA. is not maternally inherited and can be permanent. The second type of repression is due to some substance. in the luevis cytoplasm, is maternally inherited and is transient . Repression of mulleri rRNA synthesis is leaky and the leakiness seems to increase with developmental age. In some adult tissues nearly equal amounts of Levis and mulleri rRNA synthesis were found (Table 3). Cassidy & Blackler (unpublished re,sults) have found that in older hybrid embryos a small number of cells have two normal-size nucleoli, whereas the rest have only one nucleolus per cell. This suggests that the leakiness is due to the nearly normal synthesis of mulleri rRNA in a few cells rather than a small amount of synthesis in all cells. The appearance of mulleri rRNA synthesis in embryos of the l/m or m/l genotypes could be due to an escape from repression by some cells. Alternatively, it could be that a few cells are never repressed and their descendents gradually become numerous enough to be measured. The present data do not allow a decision between these two alternatives. .In embryos of the l-/m genotype almost all cells appear to be involved in the appearance of mulleri rRNA synthesis, since all cells of the embryos have no nucleolus at stage 20 but show one nucleolus per cell at the same stages as mdleri rRNA synthesis is active (Cassidy & Blackler, unpublished results). In these hybrids (l-/m) the appearance of mulleri rRNA synthesis is almost certainly due to an escape from repression because it is unlikely that almost all cells of each embryo are replaced by different clone(s) within such a short interval.

226

‘l’. HONJO

AND

R. H.

(b) Nucleolar dominance is “allelic

REEDER

repression ” of rDNA

Nucleolar dominance in Xenopus hybrids is the first instance where the phenomenon has been studied biochemically as well as cytologically. Our biochemical measurements on hybrid embryos are in agreement with t,he cytological observations of Blackler and co-workers (Blackler & Gecking, 1972; Cassidy & Blackler, unpublished results). In general, repression of mulleri rDNA correlates well with the disappearance of one nucleolus in the hybrid cells. Synthesis of both species of rRNA correlates with the appearance of two nucleoli per cell. Since laevis rDNA is essential for permanent repression of mulleri rDNA, nucleolar dominance in Xerwpus hybrids is a case of “ allelic repression ” analogous to several examples found in other genes in other hybrids (Castro-Sierra & Ohno, 1968; Klebe et al., 1970). Nucleolar dominance has also been described cytologically in Crepis hybrids by Navashin (1934). Similar cytological observations have been reported in interspecific hybrids of Salix (Wilkinson, 1944) and Ribes (Keep, 1962). It is interesting to note that only mouse type rRNA has been detected in humanmouse somatic hybrid cells (Elcieri & Green, 1969; Bramwell & Handmaker, 1971). However, it remains to be determined whether this is due to a loss of human ribosomal genes, repression of the human rDNA or aberrant processing of the human rRNA precursor. (c) Mechanism

of X. mulleri rDNA

repression

In hybrids of the genotypes l/m and m/l, repression of mulleri rDNA seems to be due to the presence of luevis rDNA. At least two possibilities may be considered as the mechanism of this repression. (1) Some product(s) of laevis rDNA functions as a repressor of mulleri rDNA transcription. (2) laevis rDNA has a higher affinity than does mdleri rDNA for some essential molecule that is in scarce supply. At this time we are unable to exclude either of these possibilities and simply point out an interesting observation by Wallace t Langridge (1971), that in certain Crepis hybrids the repressed chromosomes can resume their activity to form nucleoli during pollen formation when isolated from dominant chromosomes only by a nuclear membrane and the intervening cytoplasm. Crosby (1957) similarly found that a certain nucleolar organizer of wheat is reactivated when isolated in a micronucleus. In hybrids of the genotype l-/m, repression of mulleri rDNA seems to be due to some substance(s) in the maternal (Zuevis) cytoplasm, since there is no repression in the reverse cross (m/l - genotype). Brown & Blackler (1972) found that during oogenesis in Xenopus hybrids (l/m or m/l genotype) only Levis rDNA was amplified. Whether this gene amplification dominance in oocytes is related to the transcriptional dominance seen in somatic cells is unknown. (d) Turn-on of rDNA during early embryogenesis Previous biochemical studies (Brown 6 Littna, 1964) have shown that synthesis is not detectable in wild type laevis embryos until gastrulation at stage 10. More recent work by Emerson t Humphreys (1970,1971) on sea urchin embryos has suggested that in fact rRNA may be synthesized from fertilization onward but its detection is obscured by other RNAe before gastrulation. Our analysis of embryos from the cross bevis (l-/l) x mulleri (m/m) shows that the mixed population of embryos makes less than 5% mdleri rRNA at stage 13 to 14.

rRNA

SYNTHESIS

IN

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FROGS

227

By inference this means that the l-/m embryos are making at least tenfold less rRNA at this stage than are the l/m embryos. Between stage 13 and stage 43, mulEeri rRNA synthesis rises to its theoretical maximum, about 50% of total rRNA synthesis. We conclude that in embryos of the genotype l-/m, rRNA synthesis is regulated during early d.evelopment. The close correlation between nucleolar number and our biochemical data supports the idea that in normal laevis embryos rRNA is not synthesized in cells which lack nucleoli. Since definitive nucleoli do not appear until stage 10 in Zaevis embryos, it i:; likely that the rDNA is also turned on at that time.

APPENDIX

Measurement of laevis versz~smutleri rRNA by molecular hybridization: calculation of the standard curve The line drawn through the experimental points in Figure 1 is a theoretical curve that was derived in the following manner. Consider a single hybridization reaction which contains two filters: one with purified laevis rDNA, the other with purified mullerd rDNA bound to it. To these filters are added 3H-labeled RNA synthesized in isolated Zuevis nuclei (standard RNA) and a mixture of 32P-labeled nuclear RNA, part from Zaevis and part from mulleri (unknown RNA). A large excess of unlabeled 18 S and 28 S rRNA is also added. The following equations can be written describing the amount of 3H and 32P bound to each filter during the hybridization reaction. 3H bound to laevis filter = k,cl,

(1)

32P bound to Levis filter = k,aE + k,bl,

(2)

3H bound to mulleri filter = lc,cm,

(3)

32P bound to mulleri filter = k,bm -j- k,am,

(4)

where a = 32P-labeled Levis nuclear RNA added (cts/min), b = 32P-labeled mulleri nucleas RNA added (cts/min), c = 3H-labeled Levis nuclear RNA added (&s/mm), k, = fraction of RNA binding to homologous DNA, k, = fraction of RNA binding to heterologous DNA, 1 = amount of laevis rDNA, and m = amount of mulleri rDNA. We have assumed: (1) that the amount of hybrid formed is proportional to the RNA input and to the amount of DNA on the filter; (2) that over the range of RNA and DNA amounts used the ratio k,lk, is a constant; (3) rRNA is the same fraction of total nuclear RNA synthesis in luevis as in mulleri, since rRNA accounts for more than 90% of nuclear RNA synthesis in the presence of a-amanitin (Reeder & Roeder, 1972). By combining equations (1) through (4), we can write expressions for the 3H/32P ratio binding to the luevis illter (L ratio) and for the analogous M ratio. Dividing the L ratio by the M ratio and collecting the variables yields the following: L,Mratio=(I+K)[(i-l)(s)-kI]-‘-K,

(5)

228

T. HONJO

where K = k,lk,. Equation which is equal to the fraction

AND

R. H.

REEDER

(5) expresses the L/M ratio as a function of b/(a-i-b), of mulleri rRNA in the original [32P]RNA mixture.

When all of the 32P-labeled nuclear RNA is derived from mulleri (i.e. b/(a+b) = I.) equation (5) reduces to L/M = K2. Therefore, the value of the constant K can be determined experimentally. The average value of K in six experiments was found to be 12.6. The theoretical curve drawn in Figure 1 was calculated using equation (5) and K = 12.6. The assumptions that were made in order to write equation (5) almost certainly are an over-simplification of a complex reaction. The close agreement between the calculated curve and the experimental points suggests, however, that the assumptions are sufficiently accurate for our purpose. The most crucial assumption is that the L/M ratio is independent of the RN14 or DNA input. The data in Figure 2 show that, within the range we used, this prediction is correct. We thank Drs A. Blackler and D. Cassidy for permission to quote unpublished results, for stimulating criticism, and for providing some crucial hybrid animals. We thank Drs D. Carroll, D. Brown, I. Dawid and R. Williamson for criticism of the manuscript, Mr W. Duncan for expert histological assistance and Miss L. Costello for excellent technical assistance. REFERENCES M. L., Chipchase, M. & Speirs, J. (1971). Prog. Nucl. Acid Res. and Mol. Biol. 11, 351-389. Blackler, A. W. & Gecking, C. A. (1972). Develop. Biol. 27, 385-394. Bramwell, M. E. & Handmaker, S. D. (1971). Biochirn. Bbphye. AC& 232, 580-583. Brown, D. D. & Blackler, A. W. (1972). J. Mol. BioE. 63, 75-83. Brown, D. D. & Littna, E. (1964). J. Mol. Biol. 8, 669-687. Brown, D. D. & Weber, C. S. (1968). J. Mol. Bill. 34, 661-680. Brown, D. D., Wensink, P. C. & Jordan, E. (1972). J. Mol. Btil. 63, 57-73. Castro-Sierra, E. & Ohno, S. (1968). Biochenz. Genet. 1, 323-335. Crosby, A. R. (1957). Anaer. J. Bot. 44, 813-822. Dawid, I. B., Brown, D. D. & Reeder, R. H. (1970). J. Mol. B&Z. 51, 341-360. Elicieri, G. L. & Green, H. (1969). J. Mol. BioZ. 41, 253-260. Elsdale, T. R., Fischberg, M. & Smith, S. (1958). Exp. Cell Res. 14, 642-643. Emerson, C. P., Jr. & Humphreys, T. (1970). Develop. BioZ. 23, 86-112. Emerson, C. P., Jr & Humphreys, T. (1971). Science, 171, 898-901. Honjo, T. & Reeder, R. H. (1973). Fed. Proc. 32, 656 Abs. Keep, E. (1962). Can. J. Genet. Cytol. 4, 206-218. Klebe, R. J., Chen, T. & Ruddle, R. H. (1970). Proc. Nat. Acad. Sci., U.S.A. 66, 12261227. Navashin, M. (1934). Cytologia, 5, 169-203. Nieuwkoop, P. D. & Faber, J. (1956). Normal Table of Xenopus laevis (Daudin). North Holland Pub. Co., Amsterdam. Reeder, R. H. & Roeder, R. G. (1972). J. Mol. BioZ. 67, 433-441. Wallace, H. & Birnstiel, M. L. (1966). Biochim. Biophys. Acta, 114, 296-310. Wallace, H. & Langridge, W. H. R. (1971). Heredity, 27, 1-13. Wilkinson, J. (1944). Ann. Bot. 8, 269-289.

Birnstiel,