Size distribution and stability of DNA-like RNA synthesized during development of anucleolate embryos of Xenopus laevis

J. Mol. Biol. (1966) 19, 399-422

Size Distribution and Stability of DNA-like RNA synthesized during Development of Anucleolate Embryos of Xenopus laevis DONALD D. BROWN

Department of Embryology, Carnegie Institution of Washington Baltimore, Maryland, U.S.A. AND

J. B.

GURDON

Department of Zoology, Parks Road, Oxford, England (Received 8 March 1966) The high molecular weight RNA synthesized by anucleolate (O.nu) embryos of the toad Xenopus laevis is DNA-like in base composition. The following observations were made on dRNAt synthesized by O-nu embryos. At all stages of development, pulse-labeled RNA is very heterogeneous and 75% sediments more rapidly than 20 s ("heavy" dRNA). During development of the embryo, heavy dRNA accumulates until tail bud stage when the organ primordia have been formed. After this time, a chase of labeled RNA results in a slow size transition of dRNA, which ultimately gives rise to "light" dRNA that sediments predominantly between 10 and 20 s, The fact that this size change can occur when the amount of radioactive precursors in the pool is extremely low suggests that at least some light dRNA is derived from heavy molecules of dRNA. This transition cannot be more than 10% as efficient as the transition to stable ribosomal RNA from precursor molecules, and it takes many hours for completion. There is an inverse relationship between the size of dRNA and its stability; "light" dRNA is as stable as 4 s RNA and it accumulates to about one-fifth of the content of 4 s RNA. Stable light dRNA amounts to about 2% of the total cellular RNA in a swimming embryo.

1. Introduction Anuoleolate (O-nut) embryos resulting from a mating of heterozygous (l.nu) Xenopus laevis develop normally to the swimming tadpole stage (Elsdale, Fischberg & Smith, 1958) but do not synthesize ribosomal RNA (Brown & Gurdon, 1964). The possibility that the O-nu embryos might synthesize rRNA in any form (perhaps as precursor rRNA which is rapidly degraded) is made unlikely by the recent finding of Wallace & Birnstiel (1966). They have shown that the genetic defect is a deletion of at least

t Abbreviations used: dRNA, RNA having the same percentage guanylic-cytidylic acid content as DNA; rRNA, 288 and 18 s ribosomal RNA; "high molecular weight" RNA refers to the RNA fraction which passes through a Sephadex GIOO column without being retarded and sediments more rapidly than 108; "heavy" dRNA is RNA from O-nu embryos sedimenting in a sucrose gradient more rapidly than 20 s (see Figs I and 6); "light" dRNA is RNA from O-nu embryos sedimenting in a sucrose gradient between 10 and 208 (see Figs' I and 6); RSB, reticulocyte standard buffer, which contains 0·01 M-Tris, 0·01 M-KCl, 0·001 M-MgCl2 at pH 7·4; O-nu embryos are homozygous for the anucleolate mutation; "control" embryos include both heterozygote (I-nu) and wild-type (2-nu) embryos. The heterozygote embryos synthesize rRNA at the same rate as the wild-type embryos (Brown & Gurdon, 1964); U/C, A/G; UMP/CMP and AMP/GMP, the pyrimidine nucleotide and purine nucleotide ratios. 399

400

D. D. BROWN AND J. B. GURDON

95% of that portion of the genome which is complementary to rRNA. The apparent inability of O-nu embryos to synthesize either 28 S, 18 s, or rRNA precursor molecules presents these embryos as uniquely suited for the study of the metabolism of high molecular weight RNA which is non-ribosomal. In this study, the high molecular weight H,NA synthesized by O-nu embryos is analyzed with respect to its size distribution, base composition, stability and capacity to hybridize with homologous DNA. The results show that most pulse-labeled RNA of the O-nu mutant is "heavy" dRNA, and sediments more rapidly than 20 s. After tail bud stage, a chase of labeled RNA results in a size transition of dRNA until it sediments between 10 and 20 S ("light" dRNA). Radioactive light dRNA is as stable as labeled 4 S RNA. Finally, the origin of light dRNA has been investigated.

2. Materials and Methods Embryos were staged according to the developmental table of Nieuwkoop & Faber (1956). Mating ofheterozygotes and identification of O-nu embryos of X. laevis was carried out as described by Brown & Gurdon (1964). In all experiments reported here, O-nu embryos were isolated by phase-contrast microscopy. All embryos were frozen at - 70°C until they were processed. (a) Techniques for labeling embryos (i) 32P04

A O-nu embryo was identified at stage 26 and used as a source of nuclei for transplantation into unfertilized eggs derived from a Wild-type female (Elsdale, Gurdon & Fischberg, 1960); the eggs had been labeled with 32P0 4 (Kutsky, 1950) and their nuclei inactivated with ultraviolet irradiation. Thus, 32P-labeled O-nu embryos were obtained in which all RNA was synthesized from radioactive precursors labeled from the beginning of development. (ii) [3H]Ribonueleosides Embryos from a mating of 2 heterozygotes (l-nu) were injected with 0'03,..1. of [3H]uridine (10 elm-mole) or [3H]guanosine (2 elm-mole). At appropriate stages O-nu embryos were identified by phase-contrast microscopy and frozen. (iii) 14C02 Intact embryos were incubated with 14C0 2 according to the method of Cohen (1954) as described by Brown & Littna (1964a). (b) Isolation and characterization of RNA The techniques for isolation of whole RNA and ribosome-associated RNA from X. laeuis have been described (Brown & Littna, 1964a). A modification substit.utes one passage through Sephadcx GI00 (Brown, 1965) for the repeated precipitations of RNA with ethanol. In addition to abolishing the requirement for carrier RNA, this technique has the further advantage of separating low molecular weight contaminants and 4 s RNA from RNA of higher molecular weight (dRNA and rRNA). RNA purified by two phenol extractions was fractionated at 4°C on a 2 em X 35 em column of Sephadex GI00 which had been equilibrated with 0'01 M-sodium acetate (pH 5) containing 4 ,..g/ml. polyvinyl sulfate. The material passing through the column in the unretarded fraction and that in the next 40001. (containing the 4 s RNA) were collected separately and concentrated in vacuo wit.h a rotary flash evaporator. The unretarded material from the column was concentrated and overlaid directly onto a sucrose gradient. Tho conditions for density-gradient centrifugation and fractionation of the gradients have been reported (Brown & Littna, 1964a). The sucrose gradients contained 0·1 msrEDTA and 0·01 M-sodium acetate at pH 5; there was no polyvinyl sulfate in the gradient solutions. The EDTA and low salt concentration minimize the aggregation of different

D~A-LIKE

RNA SYNTHESIS BY

A~UCLEOLATE EMBRYOS

401

classes ofR~A which occurs at high salt concentrations (Asano, 1965). The optical density of each fraction was measured at 260 mIL and the RNA was precipitated with 5% trichloroacetic acid and caught on a Milliporo filter for counting in either a gas-flow or liquid-scintillation counter. The 4 s RNA was purified and its radioactivity was measured after fractionation on a methylated serum albumin-kieselguhr column (Mandell & Hershey, 1960); the sample for fractionation was prepared by concentrating the 40 ml. which was eluted from a Sephadex 0100 column after the unretarded fraction. This sample was neutralized to pH 7 with tris, absorbed onto a 3-g methylated serum albumin-kieselguhr column and eluted with a linear gradient of ~aCl (0,1 to 0·8 M) in 0·05 M-'l'ris (pH 7·2). Each fraction was assayed for acid-insoluble radioactive material as described for sucrose gradient analysis. Carrier RNA was isolated from ribosome pellets derived from unfertilized X. laevia eggs. The ribosome pellets were extracted at pH 5 with sodium lauryl sulfate and phenol; the aqueous phase was passed through a Sephadex column as described above. The RNA which passed through the column unretarded was concentrated in vacuo and used without further purification. (c) Base analysis The method used for nucleotide analysis of [32P]RNA has been described by Brown (1965); it has been modified slightly for use with 14C0 2-labeled embryos. Following alkaline hydrolysis of the RNA and separation of the 2',3' -nucleotides by descending paper chromatography, in Lane's solvent (1963), the radioactive nucleotides were eluted from the paper with water; the eluant was dried in liquid-scintillation vials, and the residue was dissolved in 0·5 ml. hyamine, and counted after the addition of phosphor. (d) Validity of base analysis of[uG]RNA In order to estimate the base composition of RNA labeled with 14C0 2, certain assumptions must be made beyond those normally employed for base analysis of [32P]RNA. The proportions of labeled bases will not give the actual nucleotide composition of the R~A. However, we can compare the radioactivity in pyrimidine or purine nucleotides of two types of RNA which have different nucleotide compositions if they have been synthesized from the same precursor pool. After pulse-labeling with 14C0 2, pyrimidine nucleotides are labeled almost exclusively in the C(2) position of uracil (Cohen,1954). For this reason the RNA which is synthesized just after labeling has a very high ratio of radioactive uridylic acid to cytidylic acid (Table 2). During the chase period following removal of embryos from the labeled medium, the ratio drops as uridine nucleotides are converted to cytidine nucleotides in the acid-soluble pool (Table 3). Therefore, the ratio of [14C]UMP/[14C]CMP in R~A reflects both the actual base composition of the R~A, and the extent ofthe conversion oflabeled uridine nucleotides to cytidine nucleotides in the pool. RNA which was synthesized during or shorUy after administration of the label can be distinguished from RNA of similar base composition made later by the change in the ratio of counts in the pyrimidine nucleotides. Adenine and guanine nucleotides are labeled independently by 14C0 2, and therefore any intereonversion which takes place is obscured. However, if the assumption is made that the relative 14C0 2 fixation into adenine and guanine nucleotides is the same in O-nu and control embryos, the ratio of radioactive adenine to guanine nucleotides in RNA of the O-nu embryos can be compared with that in RNA of control embryos at the same time after pulse-labeling. The interpretation ofthese base composition measurements implies the following assumption: that the relative specific activity of the two purine nucleoside triphosphates in the precursor pools is the same in O-nu and control embryos if measured in both kinds of embryos at the same time after label was administered. The same is assumed to be true of the pyrimidine nucleotides in the acid-soluble pools. Although accurate pool measurements of O-nu embryos have been handicapped by the small amount of material available, optical density measurements of the total acid-soluble fraction suggest that comparable contents of soluble nucleotides are present in O-nu and control embryos. During development, the rate of utilization of acid-soluble nucleotides will become progressively lower in O-nu embryos compared with control embryos, since rR~A synthesis which is absent

402

D. D. BROWN AND J. B. GURDON

in O-nu embryos accounts for the majority of total RNA synthesis in control embryos at later stages of development. The assumption made above is probably less valid for the ratio of specific activities of the pyrimidine nuclootidos than that of purine nucleotides, since the former ratio depends upon tho conversion of [14C]uracil nucleotides to [14C]cytosine nucleotides. The rate of this conversion may be affected by tho rate of utilization of either or both pyrimidine nuclcotides. Therefore, to increase tho validity of conclusions drawn from [14C]base ratios, we have compared the proportions of tjle labeled bases in two different classes of RNA which have widely different base compositions but which were extracted from the same embryos. In the CMe of O-nu RNA, this has been done by comparing the high molecular weight RNA (dRNA) with low molecular weight 4 8 R~A which have actual besc compositions of 40% and 60% 2',3'-OMP-2',3'-CMP, respectively (Brown & Littna, 1964a) and UMP/CMP ratios of about 1'4 and 0'7, respectively. (See 32P-labeled base analysis in Table 1.)

3. Results High molecular weight RNA synthesized by O-nu embryos has been studied by pulse-labeling embryos at different stages of development followed by various periods of chase. Experiments were performed using 32P04 , 14 0 0 2, [3H]uridine and [3Hlguanosine as radioactive precursors of RNA. Eaeh ofthese precursors has certain advantages which will be emphasized in the following sections. (a) Base composition and sedimentation pattern of [32 P] RNA synthesized by O-nu embryos In order to measure the true base composition of the high molecular weight RNA synthesized during development, 32P-Iabeled O-nu embryos were analyzed at three stages: gastrula, late neurula and heartbeat. In Fig. 1, the radioactivity profiles of RNA isolated from O-nu and sibling control [32P]embryos at stage 35 (heartbeat) are

1000 'C

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FIG 1. Sucrose density-gradient sedimentation patterns of [32PJRNA isolated from 25 O-nu and 25 sibling control embryos at stage 35. Mter ultraviolet enucleation, 32P-labeled unfertilized eggs were injected with nuclei from a single O-nu embryo (stage 26) and then allowed to develop to stage 35. Control embryos of the same stage were derived from sibling [32P]fertilized eggs. At the beginning of the extraction, 0·78 mg of carrier RNA (see Materials and Methods) was added. The R~A was purified by one precipitation with ethanol-NaCl, subjected to DNase digestion, and purified further by 2 additional precipitations. Control RNA (-e-e-) and O-nu R~A (--0--0--) are plotted together. The sedimentation pattern of [32P]0_nu RNA is plotted again in Fig. 10 (right) with tho optical density of each fraction.

DNA-LIKE RNA

SY~THESIS

BY ANUCLEOLATE EMBRYOS

403

plotted together. Base analysis from equivalent regions of these and other gradients are shown in Table 1. The base ratios of 4 s RNA are the same for the O-nu and control embryos, and the total radioactivity in the 4 s region ofthe gradient is also comparable (Fig. 1). With continuing development, the percentage 2',3'-GMP-2',3'-C:Mr content of the 32P-labeled high molecular weight RNA of the control embryos becomes increasingly ribosomal-like, but the 32P-labeled high molecular weight RNA of the O-nu embryos remains DNA-like in base composition and does not vary significantly in over-all base composition at the three stages studied. TABLE

1

Base composition of [32 P] RNA from

High molecular weight RNA

Stage of embryos

O-nu

Gastrula

o· nu and control embryos

2',3'-CMP

21

Control

GMP AMP UMP

18 34 27

Late neurula

2',3'-CMP

20

44 21 31 24

GMP AMP UMP

20 32 28

Heartbeat

2',3'.CMP

20

(35)

GMP AMP UMP

21 31 28

28

61

58 33 21 18

28

29

59

60

29 22 18

59 30 23 18

31 23 19

31

41

29

27 48

27 28 24

Control

58 30 24 18

21 40

(20)

O-nu

28

23

39 (11-12)

4sRNA

31 22 19

60

31 23 17

Each value is the average of duplicate assays. The numbers in italics are the mole per cent of each determination. DXA of X. laeois is 42% guanylic-cytidylic acid (Dawid, 1965). Acid-soluble 32PO. was present in the embryos from the beginning of development. Sucrose gradient analyses of the RNA preparations analyzed here for base composition are depicted in Figs 1 and 10. 2',3'-G~P-2',3'-CMPcontent

(b) RNA synthesized by O-nu embryos during a short pulse with

14

002

O-nu and control embryos were separated at stage 31 by phase-contrast microscopy and pulsed with 14C02 for 25, 40 and 60 minutes. Sucrose density-gradients of the purified RNA arc recorded in Fig. 2. The control embryos synthesized about five times as much high molecular weight H.NA as the O-nu embryos. Most of the newly synthesized RNA from O-nu embryos is "heavy" H.NA (greater than 20 s) with only a small amount of RNA sedimenting as "light" RNA (between 10 and 20 s). In Table 2, the nucleotide analyses of control and O-nu [14C]RNA are compared. Both the radioactive DfC and AfGratios of the O-nu RNA arc much higher than those of control H.NA. Since the independent analyses of purines and pyrimidines give the same result, 26

D. D. BROWN AND J. B. GURDON

404

Control

Control 25 min

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40 min c

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FIG. 2. Sedimentation patterns of pulse-labeled [14C]RNA. O-nu and control embryos at stage 31 (25 each) were pulsed separately with 14C02 for 25, 40 and 60 min. At the beginning of the RNA extraction, 0·7 mg of carrier RNA was added. The RNA was purified by 2 precipitations with ethanol-NaCI. The patterns of HNA sedimentation from control embryos are plotted in the upper throe graphs and those from O-nu embryos in the lower ones. O.D'280, -0-0-; radioactivity, -e-e-.

T.ABL}~ 2

[uO]Purine and [uO]pyrimidine nucleotide ratios of pulse-labeled RNA Duration of pulse (min) 25

Control O-nu O-nl~/controlt

40

Control O-nu O-nulcontrol

60

b

at

Control O-nu

O-nulcontrol

c

d

U/C

A/G

U/C

A/G

U/C

A/G

U/C

A/G

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0·59 1·6 2·7

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0·41 1·4 3·4

6·6 13 2·0

0·49 2·1 4·3

7·5 10 }·3

0·44 1·4 3·4

Jl'5 24 2·1

0·67 1·33 2·0

5·8 18·5 :J·2

0-43 1·90 4·4

6·9 17·4 2·5

0·55 1·9 3·4

6·3 Jl·O 1·8

0·50 1·53 3·1

4·5 8·8 2·0

0·62 2·1 3·4

4·0 9·4 2·3

0·4 2·3 5·7

3·2 5·5 1·7

0·31 1·6 5·2

not measured

t After counting, filters were removed from the scintillation vials and pooled according to the regions demarcated by the letters a to d (see Fig. 2, top left). The filters were freed of phosphor by rinsing in toluene, then freed of toluene by rinsing in chloroform. The filters were dried and the RNA eluted from the filters with 0·1 M.NH 40H. Conditions for subsequent alkaline hydrolysis of the RKA and separation of the 2',3'-mononucleotides have been reported (Brown, 1965). t The O-nu/control value is a ratio of the two ratios given directly above it. The values expected from [32P]base analysis for both purine and pyrimidine nucJeotides should be about 2 for either dR~A/rRNA or dRNA/4 8 RNA.

DNA-LIKE RNA

SY~THESIS

BY ANUCLEOLATE EMBRYOS

405

it is strong evidence that the true base composition of the RNA made by O-nu embryos during the pulse period has a low percentage 2',3'-GMP-2',3'-OMP content, while the RNA synthesized by control embryos at the same stage has a high percentage 2',3'·GMP-2',3'-OMP content characteristic of molecules which are precursor to ribosomal RNA. The variation is due to the few counts involved in O-nu analyses (see Fig. 2, lower part). The ratios of labeled purine and pyrimidines in RNA of both O-nu and control embryos which sediments in the 4 s region (d region of Fig. 2) are about the same as the ratios in the other parts ofthe gradients (a to c regions of Fig. 2). During these brief pulse periods, only very small amounts of radioactive 4 s RNA are synthesized; these base ratios show that some newly synthesized RNA having the same base composition as the predominant species of newly synthesized high molecular weight RNA sediments in the lighter region ofthe gradient. We do not know whether this [140]low molecular weight RNA is a degradation product of the [140]high molecular weight RNA. The sedimentation properties of pulse-labeled dRNA of the O-nu embryos are similar but not identical to those of rRNA precursor molecules which predominate in control embryos. The pulse-labeled dRNA shows a more heterogeneous size distribution than the precursor of rRNA, indicating the presence of a wide range of molecular sizes, most of which sediment more rapidly than 20 s (presumed molecular weight of greater than 106 ) . (e) Pulse-chase with

14

002

Having shown that 75% of pulse-labeled RNA is heavy, experiments were designed to study the fate of labeled dRNA during periods of chase. Embryos at stage 30 were pulsed for 1·5 hours with 140 ° 2' washed free of excess isotope, and then collected at three different times during their subsequent development. In the top of Fig. 3, the results ofthe O-nu and control experiments are plotted together. In the lower part, the O-nu values are replotted on an expanded scale with optical density for reference. In this experiment, the small amount of high molecular weight RNA is partly obscured by the large amounts of radioactive material at the top of the gradient (probably protein). Most of these radioactive low molecular weight substances were removed by passage of each RNA extract through a Sephadex column. Density-gradient centrifugation of high molecular weight RNA prepared in this way is shown in Fig. 4. A striking change in the sedimentation profiles of high molecular weight RNA takes place during the chase period. The change in sedimentation profile of [14 0 ]O-nu dRNA during a chase period is shown in Fig. 5. In this experiment RNA was purified from O-nu embryos which were collected at three different times after pulse labeling; the three sedimentation profiles are plotted together. RNase controls demonstrated that some of the radioactive material sedimenting with coefficients less than 20 s was not RNA but material which was insoluble in hot acid which contaminated the RNA extracts. The labeled contaminant (probably protein) gradually accumulated in pulse-chase experiments with 14

0 ° 2' When gastrula embryos of mixed genotypes were incubated for 3 hours with 14 0 0 and O-nu embryos were collected from the batch at different stages of develop2 ment, the size transition from heavy RNA to light RNA could not be measured directly due to the radioactive material which is not RNA that sediments coincidentally with RNA in the light region (Fig. 6). However, the RNA in the heavy and light

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Tube no. F IG. 3. Sed im ent at ion pa t t erns of L14C]R N A isolated fr om em b ry os collected at differen t t imes a fter p uls e -lab eling with 14C0 2 • Q·nu and control embryos were separate d , pulsed for 1· 5 hr at stage 30, transferred to no n labeled m edium, and p er m itted to develop for 2,5, 7 and 28 hr. At tho beginning of the RNA extraction, 0 ·78 mg of ca rrier R NA was added; the RNA was p recipit at ed 3 times w ith et h ano l- N a C!. D'Nase di gest ion w as carried ou t after tho first p recipitation. I n t he top three graphs, the r a d ioac ti vity patterns of cont rol and O-n u R X A are p lot ted together ; radioa ct iv it y, . - 0-0 - - (contro l); (O. nu ). I n the bottom three graphs the same ra dioa cti ve O-nu R KA p rofi le is p lotted on an expan de d scale along with opt ical d en sity from the sam e preparations (la rgely that of the carrier RKA) ; 0 .D . 2 6 0 , - 0--0 - ; radioact ivit y , There were 4 1, 25 a nd 24 embryos extracted at t ho 3 times , respecti vely.

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FIG. 4. Se di mentation p attern of [U C]RXA a t 3 an d 48 hr a fter labeling. Q·nu a nd control embry os we r e inc ub ated for 3 hr with 14C0 2 (from stage 22 to 24) a nd then

a llo we d t o develop in non-radioactive m edium. Embryos (26 pe r sample) were collected at 3 hr (st age 27) and 48 hr (stage 40) after the en d of the chase. RNA was ex tracted without the addition of car rie r a n d purified b y p assage through Sephadex G I 00. The R NA con t a in ed in the unretardod fr a cti on was con cent rated in vacuo and centr ifuged in a suc rose gradient. The upper two cu rves are t he p lots of O-nu RNA a n d t he lower ones of control R~A. - 0-0-, O .D· 2 60; rad ioactiv it y.

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FIG. 5. Sedimentation patterns of [l4C]RNA at various times afte r labeling with 14 0 ° 2' 14 0 0 2 for 2 hr at stage 31, t ransferred to non -radioact ive m edium, and collected at I, 7 and 24 hr after t he pulse period . No carrier RNA was added. The phenol-ext ract ed, high mo lecular weight R NA was purified prior to sucrose gradient cen trifugat ion by passage through a Sephadex GIOO column. The sed imentat ion pattern s of labeled RNA fr om 26 embryos at each time are plotted together with optical density. Optical density profiles of the three RNA pre parat ions wer e identical. T he symbols are : radioacti vi ty, upper, I hr; lower, -e-e-, 7 hr; --0- - 0-- , 24 hr ; Q.D ·260, - 0-0-. O-nu embryos were incubated with

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FIG. 6. Sedimentation pattern of ['4C]RNA from O-nu embryos which were labeled during gastrulation. O-nu gastrulae were incubated for 3 hr with 14C0 2 (from stage 10/11 to 12/13). After transfer to non-radioactive medium, 27 embryos were collected at stages 14, 20, 36, 40 and 44 (3t, 17, 40, 63 and 88 hr, respectively, after the pulse). The high molecular weight RNA was purified by phenol extraction and passage through Sephadex GI00. Before sucrose gradient centrifugation, each sample was incubated for 10 min at 20°C with 5 fLg/ml. DNase and 1 mM-MgCI2 • Radioactivity,

-e-e-; O.D· 260, -0-0-·

Ii

D. D. BROWN AND J. B. GURDON

410

regions of the gradients was recovered from the filters and measured as purified The ratio of radioactivity in heavy dRNA and light dRNA is plotted against time of chase in Fig. 7, and the change of the ratio attests to the different rates of breakdown of heavy and light dRNA. Of primary concern to us has been the origin of "light" dRNA. The [140]pyrimidine ratios from the experiment of Fig. 6 show that the majority of light [1 40]dRNA must have been synthesized after stage 20 (Table 3). This fact is demonstrated by the low radioactive U/O ratio of light dRNA compared to the high U/O ratio of dRNA synthesized before stage 20. High molecular weight O-nu RNA always has a labeled [ 14 0]2',3'.nucleot ides.

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0L--....!::-----:':,---:1=----;;f:::----;!-;:;----;;f::::'" 40 5 10 20 1 Hours ofter pulse

FIG. 7. The ratio of [14C]heavy to light dRNA after labeling O-nu embryos at the gastrula stage. All values for [14C]heavy and [14C]light RNA, except the I-hr point, are taken from the experiment illustrated in Fig. 6. The Lhr point is derived from the short pulse-labeled experiments shown in Fig. 2. The radioactivity in RNA from the heavy and light regions of the gradient has been measured by recovering RNA from filters, hydrolyzing it in 0·3 N-KOH, re-acidifying the solution and then passing it through another filter to remove acid-insoluble (alkali-stable) material. The 2',3'-nucleotides were separated by chromatography, eluted from the paper and counted. TA.BLE

3

[140]Pyrimidine nucleotide ratios of RNA synthesized by O-nu embryos after 1400Z administration at the gastrula stage Hours after end of 14C02 pulsef

3·5 17 40 63 88

[14C]2',3'-UMP/[14C]2',3'-CMP Stage

14 20 36 40 44

Heavy RNA Light RNA 2·64 1·24 0·94 1·10 0·92

2·82 1·43 0·96 0·97 0·95

4sRNA 1·97 0·96 0·66 0·64 0·67

Base analyses were performed on RNA eluted from filters as described in Materials and Methods and in the legend of Table 2. These are the results of the experiments illustrated in Fig. 6. Purification of 4 s RNA is described in Materials and Methods. t The embryos were originally pulsed with 14C02 for 3 hr between stages 10 and 13; they then developed further in non-radioactive medium.

DNA·LIKE RNA SYNTHESIS BY ANUCLEOLATE EMBRYOS

411

D/C ratio higher than that of 4 s RNA. Under conditions where the accumulated 4 s RNA has a radioactive D/C ratio of 0,67, which is the same as its true pyrimidine ratio, the accumulated heavy and light dRKA has a ratio of 0·9 to 1·1. However, the expected pyrimidine ratio for dRNA (see Table 1) is about 1·5. Our results do not distinguish between several possible explanations which could account for this discrepancy. (d) Size transition of [3H]dRNA during development after injection of eH]uridine at gastrulation

The problem of labeled contaminants that obscure the radioactive dRNA in gradients has been solved by using [3H]uridine as precursor instead of 14C0 2 • [3H]Uridine was injected into gastrulae and embryos were collected at various stages. The results of the RNA analyses are shown in Fig. 8. After removal of degraded DNA

(b)

10 hr

(0)

54 hr

(e)

94 hr46) (Stage

(Stage 40)

(Stage 22) 0·16

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800

600

400

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..••... 0

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FIG. 8. Sedimentation patterns of [3H]RNA from O-nu embryos injected with [3H]uridine at gastrulation. Control and O-nu embryos were injected with 0·03 Jll. of [5. 3H]uridine (Nuclear Chicago, > 10,000 mcjm-mole) at gastrulation (stage Ilf12) and ten O-nu embryos were separated from control embryos at 10, 54 and 94 hr after administrat.ionof the label. The RNA was purified without added carrier RNA and passed through Sephadox GIOO. RNA contained in the unretarded fraction was concentrated and treated with DKase and three-quarters of the sample was centrifuged in sucrose gradients (a to c). Carrier RNA (90 Jlg) was added to the remaining one-quartor of each sample and, following a second DNase digestion, they were passed through Sephadex again, Those sucrose gradients are presented in the lower three graphs (d to f). Radioactivity,

-e-e-;

O.D·260,

-0-0-·

D. D. BROWN AND J. B. GURDON

412

by a second Sephadex treatment (lower graphs), all acid-insoluble radioactive material is in RNA as measured by sensitivity to RNase. Between 54 and 94 hours, there has been little change in the sedimentation pattern of high molecular weight RNA. It is less heterogeneous than early pulse-labeled RNA and sediments between about 12 and 20 s with a broad peak at about 16 to 18 s. At this time all the radioactive dRNA is "light". (e) On the origin of light [3H]dRNA in O-nu embryosfollowing the injection

e

of H]guanosine Having demonstrated the change in sedimentation properties of dRNA with time, we tried to distinguish between two models which could explain the origin of light dl~NA. On one hand, both heavy and light dRNA could be synthesized independently, but the latter at much lower rates. According to this model, light dRNA would accumulate gradually during the chase, due to its stability, until it was the prevalent species oflabeled dRNA. Modell Heavy dRNA XTPP

~

Light dRRA

In this model k 1 > k 2 and the turnover of heavy dRNA is designated by the reversibility of the reaction. Alternatively (model 2), all or part of dRNA could be derived from heavy dRNA as is the case of 28 sand 18 s rRNA, which are derived from precursor molecules heavier than either of the two stable forms (Scherrer, Latham & Darnell, 1963). Model 2 k

l

XTP <= Heavy dRNA

k2

-+

light dRNA

The second model was proved for rR~A by demonstrating the transition from heavy rRNA to light rRNA at a. time when new RNA synthesis was inhibited by actinomycin D. We have studied the transition of dRNA at a time when RNA synthesis was continuing but when the radioactivity in the acid-soluble precursor pool was very low. The three precursors used in the experiments described so far l2 p 0 4, 14C02 and [3H]uridine) enter large nucleotide pools which remain radioactive throughout the life-time of O-nu embryos. Therefore, a pulse-chase experiment was performed with [3H]guanosine, since the guanosine nucleotide pool is much smaller and turns over more rapidly than eithcr uridine or adenosine nucleotide pools. Furthermore, it has been shown that there is no conversion of guanosine to adenosine over a long chase period (Gurdon & Brown, unpublished observations). The smaller pool makes possible an efficient chase of guanosine into nucleic acids (Gurdon, 1966, in the press) and permits a test of whether radioactive light-dl~NA is synthesized at a time when labeled heavy dRNA is present but when the pool of labeled precursors is low or absent. The que stion could then be posed experimentally whether "light" dRNA is derived from "heavy" dRNA, or whether it is synthesized de novo

r

DNA-LIKE RNA SYNTHESIS BY~ANUCLEOLATE_EMBRYOSJl 413

from the acid-soluble pool but at a lower rate than "heavy" dRNA. Despite the reduced utilization of nucleotides by O-nu embryos, an effective chase could be obtained by 35 hours after injection (Table 4). RNA isolated from embryos after [3H]guanosine injection at the gastrula stage was subjected to sucrose gradient centrifugation (Fig. 9). In these experiments all radioactivity in the gradients was in RNA, as shown by RNase digestion. The radioactivity of GTP in the acid-soluble pool was about equal to that of dRNA at stage 30 (Table 4). Although at two subsequent stages the radioactivity of the GTP dropped, that of dRNA remained unchanged and a size transition of dRNA took place (Fig. 9). Between 35 and 51 hours of development,

0.3

(0)

22 hr (Sloge 30)

(b)

35 hr (Slog& 35)

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FIG. 9. Sedimentation patterns of [3H]RNA from O-nu embryos which had been injected with [3H]guanosine at gastrulation. Embryos were injected with 0·03 fLl. of [8. 3H]guanosine (Nuclear Chicago, >2000 mojm-mole] at gastrulation (stage 11/12) and ten O-nu embryos were collected 22, 35 and 51 hr after injection (stages 30, 35 and 40, respectively). The phenol-extracted RNA was purified by two passages through Sephadex in the same manner as in the [3H]uridine experiment (see legend to Fig. 8), except that 0·2 mg of carrier RNA was added to each sample at the beginning of the isolation. Sucrose gradients were performed on one-third of each sample. Radioactivity, -e-e-; a.D·260,

-0-0-.

there was a decrease of radioactivity of about 100 cts/min/embryo in heavy dRNA and a concomitant increase in light dRNA of about the same amount. The increase in radioactivity in light dRNA during this period was about the same as the entire amount of radioactive GTP in the acid-soluble pool of the embryo. If we consider that for every 100 counts which might be incorporated into light dRNA from the pool, there should be approximately 500 incorporated into 4 s RNA (see Tables 4 and 5) and another 1000 counts into DNA by O-nu embryos, then it is even more unlikely that the low level of radioactivity in the pool could have been the source for the increase in radioactive light dRNA. The significance of the drop in 4 s RNA radioactivity between 35 and 51 hours (Table 4) is questionable, since other experiments have demonstrated that 4 s RNA is stable in embryos between stages 30 to 40 (Table 5 and Gurdon & Brown, unpublished work). From these considerations, we feel that the evidence strongly supports the idea that at least some light dRNA is formed from a pre-existing macromolecular precursor, perhaps the "heavy" dRNA. However, the possibility that there are low molecular weight RNA precursors of light dRNA has not been ruled out.

t> o

a: '"

D. D. BROWN AND J. B. GURDON

414

TABLE

4

Radioactivity in the dRNA, 4 s RNA, and GTP of O·nu embryos injected with [3H]guanosine during gastrulation Hours after isotope injection

Stage of embryos

22 35 51

30 35 40

Cts/min/embryo c:I.RNA/4 s RNAt GTPII

422 67 91

dRNA§ 4 s RNAt

530 550 560

2877 2798 2193

0·2 0·2 0·3

t 2 hr after [3H]guanosine injection at stage 35, the radioactive dRNA/4 s R~A ratio is 4. t Purification of 4 s R~A is described in Materials and Methods. § Cts/min in dRNA is calculated from the radioactivity in the sucrose gradients shown in Fig. 9. II [3H]GTP content of the acid-soluble pool was measured by isotope dilution. Exactly I /Lmole of GTP was added to embryos at the beginning of the R~A extraction. The nucleotide fraction was recovered from the Sephadex GIOO column in the 100 ml, eluted from the column after the collection of 40 m!. containing the 4 s RNA. The nucleotide fraction was concentrated, extracted 5 times with ether to remove phenol, then made 0·5 N with perchloric acid and passed through a MiIlipore filter. Kucleotidcs in the filtrate were adsorbed on 100 mg of acid-washed activated Norit by incubation at O°C for 15 min. The charcoal was collected, washed, and eluted with 50% ethanol containing 2% NH 3. The eluate was concentrated and chromatographed in isobutyric acid: NH 3 : H 2 0 (57: 4: 39) at room temperature for 24 hr. The ultraviolet-absorbing material representing GTP was eluted from the paper with water, the optical density was read, and the solution counted in a liquid-scintillation counter. From the specific activity of GTP in the final isolate and the known amount of excess GTP originally added (l/Lmole). the radioactivity originally present in GTP could be calculated at each of the three developmental stages analyzed. Endogenous GTP (in /Lg) is negligible compared to the added carrier GTP.

(f) Absence of light dRNA in early embryogenesis

The actual content of dRNA increases in embryos as they develop (Brown, 1965). Embryos which were injected at gastrulation with [3H]ribonuelcosides contain mainly heavy dRNA ten hours after labeling (Fig. 8). The absence of any increase in the amount of light dRNA until after stage 20 (neurula) is shown in [32P]0_nu embryos in which all the RNA synthesized during development is radioactive. [32P]RNA was purified at three stages of development and analyzed by sucrose gradient centrifugation (Fig. 10). Centrifugation time was prolonged for the RNA from the early stages so that [32P]RNA in the light region of the gradient could be visualized. Much of the heavy dRNA has sedimented to the bottom of the tube under these conditions; yet only a small amount of [32P]RNA from gastrula and neurula embryos sediments in the region of light dRNA. By stage 35 most dRNA is light. (g) Stability of dRNA

There appears to be an inverse relationship between the size of dRNA and its stability (Fig. 7). The heaviest species labeled during a short pulse are degraded most rapidly, whereas the smallest sizes ("light"-l0 to 20 s dRNA) are as stable as 4 s RNA (Tables 4 and 5). Once the chase has proceeded to the point that the predominant size oflabeled dRNA is "light", there is no change in the amount of this labeled RNA relative to labeled 4 s RNA. Eventually, the radioactivity in both classes of RNA (as well as the maternal rRNA) begins to decrease in the O-nu embryos, but only after their development is arrested (see Table 5).

DNA-LIKE RNA SYNTHESIS BY ANUCLEOLATE EMBRYOS

Gastrula (11/12)

6

Neurula (20)

80

415

Stage 35

400

160

c

'E

60

r

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.•...

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10

20

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.,e'•.•.•.

10

20

o

j

~

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Tube no.

FIG. 10. Sucrose gradient sedimentation of [32P]RNA of O-nu embryos at early and late stages of development. Since these embryos were derived from transplantation of O-nu nuclei into 32P-labeled eggs (see Materials and Methods), all RNA synthesized during development was labeled. Since different batches of 32P_labeled eggs were used for each analysis, the amount of radioactivity in the three RNA preparations cannot be compared. After phenol extraction, RNA was purified by ethanol precipitation and DNase treatment; 0·78 mg of carrier RNA was added to the initial homogenate. Longer centrifugation (18 hr) of the RNA from early embryos (stages 11/12 and 20) further resolves the radioactive profile of RNA sedimenting in the region of light dRNA. The base analysis of RNA from this experiment is reported in Table 1. The gradient of O-nu RNA from stage 35 embryos (right) is the same as that in Fig. 1, but in this case it is plotted with the optical density of the preparation. Radioactivity, O.D· 260, -0-0-.

-e-e-; TABLE

5

Radioactivity in the dRNA and 4 s RNA of O-nu embryos injeded with [3H]uridine during gastrulation Hours after isotope injection

Stage of analysis

5 10 54 94

15 22 40 46

dRNA 4sRNA (cts/min/embryot)

353 597 410 233

710 1830 2260 1260

:5 0:::

/

100

~

0

. ... .

~ )

80

dRNA/4s RNA

0·5 0·3 0·2 0·2

Sedimentation profiles for 3 of the 4 RNA preparations analyzed here are depicted in Fig. 8. t Values for ctsjmin of dRNA and 4 s RNA were obtained in the same way as in the [3H]guanosine experiment. See legend of Table 4 for further details.

(h) Appearance of radioactive RNA in association with ribosomes

In normal development of X. laevis, some of the dRNA synthesized after late cleavage can be found in association with the ribosomes (Brown & Littna, I964a). Isolation of RNA at neutral pH results in degradation of the dRNA, so that it sediments with s values lower than 18 s. The association of [14C]RNA with ribosomes of O-nu and control embryos is shown in Fig. II. The sucrose gradients were performed

FIG. 11. Sedimentation patterns of [l4C]RKA associatod with ribosomes of O-nu and control embryos. O-nu and control embryos were incubated with l4C0 2 for 1·75 hr at stage 30, and 25 embryos were collected at 2·25 and again at 14·25 hr after the end of the pulse. Embryos were homogenized in 6 ml. of RSB medium containing 0·25% deoxycholate and the homogenates centrifuged at 10,000 g for 10 min. The supernates were diluted to 11 ml, with RSB medium and centrifuged at 105,000 g for 2 hr. The high-speed pellets were extracted for RNA after the addition of 0·5 rng of carrier R~A. The RNA was purified by 2 precipitations with ethanol-sodium chloride. The upper two curves are sucrose gradient profiles of O-nu RKA and the lower ones those of control RNA. Radioactivity, O.D·260, - 0 - 0 -

-e-e-;

on RNA purified from ribosome pellets. It must be emphasized again that in 14COll pulse-chase experiments there is a considerable amount of HC-Iabeled protein which is extracted with the RNA, and in this case sediments in the 4 s region. Less than half of the radioactive material in the 4 s peak in Fig. 11 (right) is RNA. An unsuccessful attempt was made to isolate RNA from "nuclear" and "cytoplasmic" fractions of homogenates. Degradation of RNA occurred after only four minutes exposure to neutral pH at OcC in the absence of sodium lauryl sulfate. (i) Hybridization 0/ RNA/rom O-nu and controlembryos with X.laevis DNA One property of dRNA which distinguishes it from rRNA and 4 s RNA is the relatively small amount of DNA required to hybridize large proportions of dRNA. This is due to the small number of dRNA molecules in a cell that are complementary to a given stretch of DNA. An obvious prediction is that all high molecular weight RNA synthesized by the O-nu embryo should be readily hybridizable. Several questions

D~A-LIKE

RNA SYNTHESIS BY ANUCLEOLATE EMBRYOS

417

were of interest in these experiments. First, would all the RNA be hybridized at relatively low "inputs" of DNA? Second, would the extent of hybridization of "heavy" and "light" dRNA show different patterns? The experiment was designed with the hope of detecting differences between pulse (heavy) dRNA and chased (light) dRNA in the number and variety of copies they contain. Therefore, the same solution of RNA was repeatedly incubated with fresh DNA filters. Hybridization should withdraw RNA molecules having few copies per complementary stretch of DNA more efficiently than those (such as rRNA) which are present in large numbers of copies per complementary region of DNA. The RNA remains acid-insoluble for a week of repeated incubation with DNA filters (though getting gradually smaller in molecular weight) and significant portions continue to be hybridized with the DNA adsorbed on each successive filter. The results of this experiment are shown in Fig. 12. Although only 12% of the high molecular weight RNA from 12

-c ~

:'§ .0;>. 8 s:

«:

z

u::

I

'"' -' "0

4

~ ;j!.

200

400 p.g DNA

FIG. 12. Hybridization of O-nu and control high molecular weight [3H]RNA with X.laevia DNA.

[3H]RNA was purified from O-nu and control embryos at stage 35. The embryos were labeled either at gastrulation or at stage 34, 2 hr before collection by injection of [3H]uridine. RNA from each of the four batches of embryos (pulsed or chased, O-nu or control) was purified after the addition of 0·3 mg of carrier RNA. After phenol extraction, RNA in the aqueous phase was precipitated once with ethanol-NaC!. The RNA was dissolved in 2 ml. of 0·1 ll£·sodium acetate (pH 5) with 4 ",g/ml. polyvinyl sulfate, and incubated 15 min at 22°C with 20 fLg/ml. DNase and 2 mM-MgCIaThe RNA solution was made 0'5% with sodium lauryl sulfate, shaken twice with phenol, passed through Sephadex and the material contained in the unretarded fraction concentrated for hybridization. Portions of [3H]RNA were hybridized to erythrocyte DNA of X. laevi8 (Dawid, 1965) immobilized on HA Millipore filters (100 fLg DNA/filter) by the technique of Gillespie & Spiegelman (1965). Prior to hybridization, each solution contained 85 fLg of RNA (75 ug derived from the carrier rRNA, 10 fLg from the embryos) in 2 ml, of 0·3 ll£-NaCl, 0·03 M-sodium citrate (2 x SSC). Each RNA solution was incubated repeatedly for 24-hr periods with new fillers each containing 100 fLg DNA. Following incubation, each filter was removed and treated with RNase as described in the original method, and then counted in a liquid-scintillation counter. Background binding ("noise" level) correction was made by incubating a filter containing the same amount of chicken erythrocyte DNA simultaneously in the RaIIl& bottle with the filter containing X.latwis DNA. LeBS than 10% of the radioactive material bound to X. laevia was bound to chicken DNA under the same conditions, and this value was subtracted as background. The curves shown here are cumulative curves in which the radioactive material (cts/min) of RNA hybridized during each successivo in cubation as well as the DNA used is added to the previous valuo. - 0 - 0 --, Pulse-labeled O-nu RNA; --0--0--, chased O-nu RNA; - . - . - , pulse-labeled control RNA; --.--.--, chased control RNA. These RNA solutions contained 1740, 1520, 6150 and 7400 cta/min, respeotively, at the beginning of the experiment.

418

D. D.

BROW~

AND J. B. GURDON

O-nu embryos eventually was hybridized under these conditions, the results in Fig. 12

are significant when the four preparations of RNA are compared with each other. Labeled O-nu dRNA from both pulsed and chased embryos hybridize with DNA more readily than control high molecular weight RNA. The hybridization curves demonstrate a slight (but reproducible) difference between pulse-labeled RNA of the O-nu embryos (heavy dRNA) and the labeled RNA still remaining after the long chase period (light dRNA). As expected, RNA from control embryos hybridized to a lesser extent after a long chase when large amounts of radioactive rRNA have accumulated.

4. Discussion (a) Evidence that O-nu embryos do not synthesize rRNA

It is concluded that all high molecular weight RNA synthesized by the homozygous O-nu mutant is non-ribosomal in character.The mutation represents a deletion of at least 95% of the portion ofthe genome complementary to rRNA (Wallace & Bimstiel, 1966). Even if the deletion is not complete, there is convincing evidence that neither rRNA nor its precursor molecules are synthesized by the O-nu embryos. Even pulselabeled RNA of O-nu embryos is DNA-like in base composition (Fig. 2 and Table 2). It is important to distinguish stable dRNA (light dRNA) from 18 s rRNA. This light dRNA is more heterogeneous than 18 s rRNA in size distribution (Figs 8 and 9), has a higher U/C ratio than that expected for rRNA (Table 3) and hybridizes more readily with homologous DNA than docs rRNA (Fig. 12). Furthermore, when the RNA associated with ribosomes is extracted under conditions where 18 s rRNA is not degraded, all the dRNA (light or heavy) is degraded to lower molecular weight molecules (Fig. II). (b) Size transition and stability of dRNA during development

For simplicity we have divided dRNA from O-nu embryos into two classes: "light" and "heavy". This classifies the heterogeneous dRNA arbitrarily by virtue of its sedimentation coefficients (see Figs 1 and 6). "Light" dRNA includes RNA sedimenting between 10 and 20 s, while "heavy" dRNA refers to RNA which sediments more rapidly than 20 s. In short pulse-labeling experiments (Fig. 2), about 75% of the labeled O-nu dRNA is heavy, with presumed molecular weights greater than one million. Other experiments not reported here show that pulse-labeled dRNA is heavy at all stages from gastrula to swimming tadpole (8-60 hr of development). A high molecular weight fraction of pulse-labeled RNA isolated from L-cells has been shown to hybridize readily with homologous DNA (Perry, Srinivasan & Kelley, 1964). The actual content of dRNA increases in the embryos as it develops (Brown, 1965). Up to the tail bud stage (25), the dRRA that accumulates is mostly heavy dRNA (see Figs 8 and 10). After stage 30, the presence of radioactive light dRNA can be demonstrated by seven hours after the beginning of a chase period (see Fig. 5). If light dRNA is synthesized de novo by embryos, it must be synthesized largely after stage 30. On the other hand, if light dRNA is derived from heavy dRNA, then those heavy molecules which are the precursor molecules for light dRNA must have been synthesized after stage 20, since the ratio U/C of the light dRNA is low (Table 3). Therefore, very little of the heavy dRNA made before stage 20 is stabilized as light dRNA. This agrees with the hybridization studies of Denis (I965), who determined

DNA-LIKE RNA SYNTHESIS BY ANUCLEOLATE EMBRYOS

419

the half.life of RNA in embryos of wild-type X. laevislabeled at early and late stages of development. At early stages, that fraction of radioactive RNA which hybridized readily with DNA decayed as a uniform population of molecules with a half-life of about two hours; there was no evidence of a stable fraction. However, the same fraction of labeled RNA from older embryos decayed with complex kinetics, indicating a mixture of molecules which had widely different half-lives, some as long as 45 hours. In the O-nu mutant, the stability of dRNA is inversely related to its sedimentation coefficient, since the heaviest molecules are degraded most rapidly and the lightest are most stable (Figs 4, 5, 7, 8 and 9). No specific half-life can be assigned to heavy dRNA from these studies; however, light dRNA, which is heterogeneous like heavy dRNA, is as stable as the 4 S RNA synthesized at the same time. After a two-hour pulse of O-nu embryos, most of the newly synthesized dRNA is heavy, and the ratio of total radioactivity in dRNA to that in 4 s RNA is about four. As the chase proceeds and most of the heavy dRNA is degraded, this ratio drops to 0·2 and does not change subsequently (Tables 4 and 5). After long chase periods, a large proportion of the dRNA is light. During this same period, there is apparently no destruction of the ribosomes which were present in the unfertilized egg, just as has been shown in wild-type embryos (Brown & Littna, 1964b). However, arrested O-nu embryos (after stage 40) gradually degrade radioactive dRNA and 4 s RNA (Table 5) as well as some of their non-radioactive ribosomal RNA. Although a normal embryo begins development with a small amount of 4 s RNA compared to rRNA, there is a gradual increase in the 4 s RNA content until in the swimming embryo it constitutes about 10% of the total RNA (Brown, 1965). If the ratio of light dRNA to 4 s RNA is 0·2 (Tables 4 and 5), the relative amounts ofrRNA, 4 s RNA and dRNA are approximately 90, 10 and 2, respectively; therefore, light dRNA represents about 2% of the total RNA of the swimming embryo. (c) Origin of light dRNA

The elucidation of the origin of light dRNA is complicated further by its slow accumulation after a pulse label. However, after injection of [3H]guanosine, accumulation oflabeled light dRNA (Fig. 9) occurred with conservation of radioactive material (counts) in the total dRNA of the embryos. During this period, counts in GTP of the soluble pool were reduced to 10 to 20 %of those in the total dRNA, an amount equal to the increased number of counts which had accumulated in light dRNA. Since most of the small amount of radioactive material remaining in the GTP pool would be expected to be incorporated into DNA and 4 S RNA rather than light dRNA, the accumulation of radioactive light dRNA in this experiment cannot be accounted for by synthesis from labeled nucleotide precursors of the acid-soluble pool. The alternative hypothesis states that at least part of the light dRNA is derived from macromolecules (Samarina, 1964; Scherrer and Marcaud, 1965). We suggest that some heavy dRNA is precursor for light dRNA. If all of the light dRNA is derived from heavy dRNA, then not more than a small proportion of heavy dRNA synthesized in a pulse label undergoes this conversion. This can be shown by comparing the appearance of light dRNA in O-nu embryos with the conversion of ribosomal precursor RNA to mature rRNA in control embryos. The O-nu embryos synthesize about 20% as much high molecular weight RNA in a pulse period as control embryos (see Fig. 2). From this we assume that control embryos make rRNA : dRNA in the ratio of 80: 20 during a pulse label. If all heavy dRNA was 27

420

D. D. BROWN AND J. B. GURDON

stabilized (transformed to light molecules) with the same efficiency as rRNA, then the embryo would ultimately contain 25% as much light dRNA as rR~A. However, since light dRNA constitutes only about 2% of the total RNA of swimming embryos, no more than 10% of heavy dRNA can be transformed to light dR~A. Therefore at least 90% of the pulse-labeled dRNA is not stabilized but degraded. The fact that conservation of radioactive material in total dRNA occurs during this transition presents a paradox, since 90% of pulse-labeled (heavy). dRNA is degraded and never gives rise to light dRNA. However, the paradox can be resolved by assuming that heavy dRNA is not transformed randomly to light dRNA, but rather that there are two distinct classes of heavy dRNA-one which is transformed to light molecules (about 10%) and one which is degraded rapidly (90%). Accordingly, after 22 hours of chase (Fig. 9,left), only the first class of heavy dRNA would remain labeled; the other 90% would have been degraded. This remaining heavy dRNA then would be efficiently converted to light dRNA during subsequent development. The idea of two classes of heavy dRNA need not imply that they are copies of different regions of the genome. This idea would have benefited from striking differences in the hybridization properties of light and heavy dRNA. The differences which were found (Fig. 12), although reproducible, are not large enough to interpret with any confidence. (d) Cellular location and function of heavy and light dRNA Attempts to establish the cellular location of heavy and light dRNA in the O-nu embryo have been unsuccessful (Fig. II) due to the extensive degradation of dRNA which takes place in neutral homogenates. However, heavy dRNA must be synthesized in the nucleus, since Wallace (1966) has shown that radioautographic localization of silver grains occurs exclusively over nuclei of O-nu cells after short exposures to [3H]uridine (up to one hour). This technique is not suitable for studying transfer of dRNA.to the cytoplasm, since there is a gradual accumulation of labeled 4 s RNA during a chase period. Moreover, it has not been possible to determine directly whether dRNA is transferred from nucleus to cytoplasm as high molecular weight molecules (heavy dRNA) or as light dRNA. It is known, however, that a large percentage of dRNA in early embryos can be isolated in association with ribosomes (Brown & Littna, 1964a). At these early stages, the dRNA is mostly heavy (Fig . 10). Whereas most workers have found that polysome-associated "messenger" RNA sediments from 8 to 12 s, recent experiments have shown that heterogeneous "messenger" RNA with much higher sedimentation coefficients can be isolated from polysomes if greater precaution is taken to reduce degradation (Penman, 1966; Nemer & Infante, 1965). Light dRNA may function as "messenger" RNA, since its sedimentation properties are virtually identical with the RNA isolated from liver and reticulocytes, which is most effective in stimulating protein synthesis in vitro (DiGirolamo, Henshaw & Hiatt, 1964; Brawerman, Biezunski & Eisenstadt, 1965). Although some heavy dRNA may be "polyeistronic messenger" RNA, the data described here are also consistent with Harris's contention (1963) that a considerable portion ofpulse-labeled RNA is degraded before leaving the nucleus (see also Edstrom, 1964). The data on dRNA presented here supports the following correlation between size transition, cellular location, and function as messenger RNA. MostdRNA is transcribed in the nucleus as polycistronic (or polygenic) molecules. (For the average gene a 200,000 molecular weight polynucleotide codes for a polypeptide of molecular weight

DNA-LIKE RNA SYNTHESIS BY

A~UCLEOLATE EMBRYOS

421

20,000.) At early stages of development, dRNA is heavy and either remains in the nucleus, where much of it is later broken down, or becomes associated with ribosomes as a polycistronic messenger RNA. When the embryo has completed its early differcntiation and the organ primordia have been established (stage 25), part of the heavy dRNA appears to be converted to stable light dRNA. Because this transition is slow and occurs hours after pulse labeling, it may take place in the cytoplasm in association with ribosomes. Many tissues go through a period of rapid cell division prior to differentiation. The beginning of cytodifferentiation and formation of tissue-specific proteins generally occurs after cell division has slowed or ceased. During the period of cell proliferation, actinomycin D prevents the subsequent appearance of specialized products of the cell (Wessels & Wilt, 1965; Wilt, 1965; Kirk, 1965). Mtcr cytodifferentiation begins, the cells become actinomycin-resistant, implying that specific messenger RNA's have become stabilized and that their further synthesis is not necessary. The transition from labile to stable messenger RNA may require a size transition like that which appears to take place in the dRNA of O-nu embryos. We are grateful to Drs 1. Dawid and C. 'Weber, Mr R. Hallberg and Mrs M. Schwartz for their critical reading of this manuscript. J. B. G. gratefully acknowledges a research grant from the Medical Research Council.

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