gene expression in amphibian development

J. Mol. Biol. (1966) 22, 269-283

Gene Expression in Amphibian Development

I. Validity of the Method used: Interspecific and Intraspecific Hybridization between Nucleic Acids. Properties of Messenger RNA

Synthesized by Developing Embryos

HERMAN DENIS?

Carnegie Institution of Wa&ingtm, Department of Emb yology, Bal&wre, Maryland, U.S.A.

(Received 24 March 1966, and in revised form 1 Au.gu& 1966)

The properties of the hybrids formed between labeled RNA from Xenqua &z.e& embryos and DNA trapped in agar have been studied. Some characteristics of the duplexes formed under the same conditions between DNA fragments and DNA in agar are also reported. DNA-DNA as well as DNA-RNA duplex formation is a species-dependent process. The melting point of the DNA-DNA and of the DNA-RNA duplexes formed at 60°C is about 16’C lower than that of native DNA. It is concluded that some inaccuracies in base-pairing exist in the hybrids resulting from 80 incubation at 6O’C. More inaccuracies are included in the duplexes formed at lower temperatures.

In embryos exposed to carbon-14 dioxide for one hour, 12 to 14% of the labeled RNA hybridizes readily with DNA. Hybridized RNA has a base composition similar to that of DNA and turns over more rapidly than non-hybridized RNA.

1. Introduction The mechanism by which the genetic information is transferred from DNA to protein is now fairly well understood. The messenger hypothesis originally formulated for bacteria (Jacob & Monod, 1961) has been successfully applied to differentiated cells of multicellular orgsnisms (Marks, Burka & Schlessinger, 1962; Scherrer, Latham t Darnell, 1963; Di Girolamo, Henshaw & Hi&t, 1964). It remains to be explained in the light of the messenger hypothesis how the inform&ion enclosed in one single copy in the fertilized egg is used to build up the many different cell types of the adult organism. It seems likely that the type of message transcribed is different in each region of the early embryo, thus causing the appearance of tissue-specific proteins. Since the tissues do not differentiate synchronously in embryonic development, it can be expected that the growing embryo contains messengers specific for each stage. The work reported here was undertaken to test this hypothesis. It will show that different regions of the genome are indeed expressed in embryos of different stages.

The hybridization technique between DNA and DNA, and DNA and RNA offers a mesus to determine the degree of homology between a nucleotide sequence taken

t Present address: Laboratoire de Biochimie, Univerait6 de Liege, 17 place D&our, Liege, Belgium.

269

270 H. DENIS

as reference and other nucleotide sequences. In these experiments, particular use has been made of the capacity of homologous non-radioactive RNA or DNA to compete with the binding of radioactive RNA or DNA to DNA entrapped in agar. The validity of this competition technique (Hoyer, MacCarthy & Bolton, 1964), as a means of comparing the messengersi synthesized by embryos of different stages, will be the subject of this paper. Several properties of mRNAS synthesized by embryos of different stages will be also described. The rate of labeling, the base composition and the decay rate of mRNA will be studied.

2. Material and Methods Unless otherwise stated, the South African aquatic toad Xenopus laevis has been used

in all the experiments described below. Developmental stages were numbered according to the tables of Nieuwkoop t Faber (1956).

(a) Breeding of anintala and collection of embryos The animals were handled and bred as described by Brown & Littna (1964). Soon after

laying, the eggs were dejellied and stripped from adhering debris by a procedure derived from that of Townes (1953). The egg masses were shaken for 2 min at 23°C in a 2% cysteine-HCl, 0.2% papain (Nutritional Biochemical Corp.) solution, adjusted to pH 8 (Dawid, 1965). The eggs were then carefully rinsed in water and raised in dechlorinated tap water.

(b) Sucrose density gradients Centrifugation in sucrose density-gredients (6 to 20%) was performed as described by

Brown t Littna (1964). The gmdients were usually spun at 6 to 6°C for 18 hr at 24,000 rev./min.

(c) Extraction and labeling of DNA DNA was extracted either from liver nuclei or from erythrocytes by a modification of

Mrtrmur’s technique (1961). Deproteinization was carried out by two shakings with chloroform followed by several shakings with phenol. In some cases, DNA was prepared by the pronase method of Berns & Thomas (1965), with equally good results. DNA from species other than X. Zuewis was extracted from livers as described above. Calf thymus DNA and salmon sperm DNA were commercial preparations.

Radioactive DNA wss obtained from whole tadpoles (stage 42), which had been labeled during earlier development with 14COa. Four pulses of 1 hr were given at 24-hr intervals (i.e. at stages 12,26 to 28,33 and 39) according to the procedure described by Cohen (1954) and Brown & Littna (1964). The DNA prepared under these conditions had a specific activity ranging from 200 to 700 cts/min/pg with a protein contamination of less than 2%. DNA of higher specific activity was obtained from tissue cultures derived from adult kidney and labeled with tritirtted thymidine (gift from Dr I. B. Dawid). This DNA had 8 specific activity of 60,000 cts/min/pg.

(d) Shearing and denaturation of DNA As shown by MacCarthy & Bolton (1963), single-stranded DNA does not renature with

DNA in agar, unless it has been fragmented to pieces of small molecular weight. DNA was fragmented by sonic&ion with a Branson microprobe (setting no. 6) for 6 min at 0°C. The preparation had been previously flushed with nitrogen for 5 min in order to reduce possible oxidation. DNA treated in this way had 8 sedimentation coefficient of about 10 s in sucrose gradient.

Denaturation of DNA was carried out by heating for 5 min at 95 to 100°C in 0.1 x SSC (0.015 ~-N&c1-0-00~5 M-sodium citmte) and rapid cooling.

t The word “ messenger ” is used here without functional implications. In these experiments, messenger is charrecterised only by its ability to hybridite with DNA.

$ Abbreviations used: mRNA, messenger RNA; sRNA, soluble RNA; rRNA, ribosomal RNA; C, cytosine; A, adenine; G, guanine; U, uracil; T, thymine; T,,,, melting temperature.

GENE EXPRESSION IN AMPHIBIAN DEVELOPMENT 271

(e) Labeling and eztruction of RNA

Embryonic RNA was labeled by injecting 1 mc of 3aP as inorganic phosphate into the female before ovulation was induced (Kutsky, 1950). Under these conditions, a large ctmount of3aP enters the inorganic pool of the fertilized egg and persists through embryonic development (Brown & Littna, 1964). All nucleic acids synthesized from fertilization to the feeding stage are thus labeled. For shorter labelings, the method described by Brown & Littna (1964) was used. The embryos were exposed to 14C0, in a tightly closed vial, half-filled with boiled salt medium, buffered at pH 6 with 0.1 ~-sodium phosphate and containing O-1 mg penicillin and streptomycin/ml. Unless otherwise stated, the time of labeling with 14COa was limited to 1 hr.

RNA W&B extracted from embryos by the sodium dodecyl &f&e-cold phenol method of Brown & Littna (1964). Most of the time, the DNase step of Brown t Littna WS,S

omitted, but two successive phenol shakings were used in order to reduce the amount of protein contamination. After a 1-hr pulse in 14COz, the labeled RNA preparations contained about 6% of its radioactivity as protein and from 0 to 6% as DNA, according to the stage. RNA continuously labeled with 3aP contained approximately the same amount of contaminants.

(f) Determination of base composition The base composition of [32P]RNA was determined after hydrolysis in O-3 M-KOH at

37°C during 18 hr. The method used is described in detail by Brown (1965).

(g) Hybridization experiments Embryo RNA ww hybridized with DNA extracted from adult tissues (blood or liver).

The use of adult DNA to hybridize RNA from embryos is justised, since preliminsry experiments have shown no detectable differences between the nucleotide sequences of embryo DNA on one hand and DNA from various adult tissues (blood, heart and liver) on the other hand. Furthermore, a substantial portion (about 75%) of DNA from adult cells can be hybridized with embryo DNA when the latter is present in large excess.

Denatured DNA prepared from adult tissues was immobilized in agar (Bolton & Mac- Carthy, 1962). The &al concentration of DNA in &g&r ranged from 0.4 to 1.3 mg/g. The hybridization tests were carried out as follows. The desired amount of RNA or denatured DNA was incubated at 60°C with DNA-agar in 2 X SSC. As already shown by Hoyer, MacCarthy & Bolton (1963), it was found that the amount of radioactive material remaining bound to DNA-agar after incubation at 60% reaches a maximum after 24 to 40 hr and depends on the volume of solution relative to the amount of agar. In view of these preliminary tests, the following conditions were adopted for both DNA-DNA and DNA-RNA hybridization? experiments. For convenience, the incubation time was limited to 15 to 18 hr. In each incubation mixture, 1 ml. of 2 x SSC was added for each gram of DNA-agar.

At the end of the incubation time, the DNA-agar was transferred to a “tea bag” (MacCarthy & Hoyer, 1964) and washed with 10 rinses of 10 ml. of 2 x SSC at 60°C (IO min each), in order to remove the unbound material. From 5 to 15% of the DNA originally present in the agar was lost by washing at 60°C. The hybridized fragments were then released from the entrapped DNA by 5 rinses (10 ml. each) in 0.01 x SSC at 75°C.

Acid-insoluble radioactive material w&9 measured by passing the content of each fraction through Millipore filters after adding 200 pg of carrier (yeast) RNA and I ml. of 55% trichloroacetic acid to each tube. The Millipore filters were then dried and counted either in a gas-flow counter or in a liquid-scintillation spectrometer.

(h) Non-specific binding The amount of non-speci& binding due to incomplete removal of free RNA or DNA

from the agar w&s measured by incubating radioactive DNA or RNA with empty agar. When DNA of high spectic activity w&8 used, the background was usually rather high, unless yeast RNA (100 to 200 pg) was added to the incubation mixture, When this pre-

t For convenience, the term “hybridization” has been used for duplex formation not only of RNA with DNA in agar, but, also of DNA fragments with DNA in agar.

272 H. DENIS

caution was taken, the amount of non-specific binding never exceeded 2.6% of the input radioactivity per g of agar, and was roughly proportional to the weight of agar used. In most experiments, the amount of DNAagar used was limited to 100 mg. In each experiment, the non-specific binding determined in parallel runs with empty agar was sub- tracted from the experimental results.

3. Results

(a) Species-dependence of the DNA-DNA and DNA-RNA hybridizations

If the hybridization between DNA in agar and RNA or DNA fragments is due to accurate base-pairing, no cross-reaction should be observed between the nucleic acids belonging to unrelated species. Some hybridization can be expected between nucleic acids of closely related organisms (Hoyer et al., 1964). In order to test this hypothesis, labeled DNA fragments from X. la&s were made to compete with increasing amounts of non-radioactive DNA from other species. If the heterologous DNA’s have nucleotide sequences in common with X. Levis DNA, they will compete for the same sites of the entrapped X. la&s DNA, and the amount of labeled fragments which hybridize with the entrapped DNA will be reduced. When unlabeled DNA from X. luevis was used as a competitor, a dilution curve was obtained which was fairly linear, when plotted on a double logarithmic scale (Fig. 1). Non-radioactive DNA from two different species of frogs (Rana @(piens and Rana sylvatica), from a salamander (Ambly&ma tigrinurn) and from a newt (Triturus viridescens) was

30

20

IC

c

6 .- ‘;; G 2c 25 I”

at IC

r

1

n -

1

I I I 5 IO 50

Portions of [“CIDNA added

FIG. 1. Species-dependenoe of DNA hybridization.

Five pg of [I’C]DNA fragments (3500cts/min) were incubated with 100 pg of DNA-war and increasing amounts of non-radioaotive DNA fmm the same species (--O-O-) or from other species: R. @piem (---@-a-), R. 8ylvaticu (-A--A-), T&m tit-i&?SOem (-u-u--L Amb@@nna tigrimmn (-A-A-), and E. coli (-a-m-). The figures shown on the ordinate refer to the percentage of input radioactive material remaining bound to DNA-agar at the end of the incubation.


much less competitive than X. Zumis DNA. Escherichia coli DNA does not compete at all with X. laevis DNA. In another series of experiments, DNA from other classes of vertebrates (mammals, birds and fishes) showed 10% or less competition for hybridization of X. laevk DNA fragments. Many of these heterologous hybridizations have been performed in the reverse direction. In these experiments, DNA from various species was entrapped in agar and hybridized with radioactive X. luevis DNA fragments.

The data are summarized in Table 1. The figures of the first column correspond to the extent to which the heterologous DNA can compete for hybridization of X. la&s DNA with X. Iaevis DNA in agar. These figures are thought to reflect the percentage of nucleotide sequences shared by the DNA of the two species (Hoyer et al., 1964). The data presented in the second column (reverse crossing) were obtained by hybridization of X. la&s DNA fragments with heterologous DNA entrapped in agar.

TABLET

Extent of cross-section between X. laevis DNA and DNA from other species

Species Direct crossing Reverse crosaing$

( y0 inhibition of (O/J of homologous reaction) reaction)

Rana p&p&ma Ram ~ylvatica Amblyhmua t@+wn Ttitu~ue virideecsne Salmon ox Hen Eschtik WEi

W 26 22t -

w 4 14t - w 11 63 4 2$ 5 @t 0

t D8t8 taken from the experiment reported in Fig. 1. $45 rnH of labeled X. kzevia DNA (2260 ots/min) were hybridized with 28 pg of X. Zaevis DNA

in agar in the presence of increasing &mounts (up to 225 rcg) of unlabeled DNA fragments from salmon, ox or hen.

8 Five m of labeled X. Latvia DNA (3600 cts/min) were hybridized with 100 M of DNA in agar of the speoies listed in the first column. Under these conditions. 28% of X. kzevis DNA fragments hybridize with X. kavis DNA in agar.

The results obtained in both types of crossings are similar. A discrepancy was, however, observed in the crossings between urodele DNA and anuran DNA. The sequences common to both urodele (salamander and newt) and anuran (frog and toad) DNA’s represent a shorter length of the urodele genome than that of the anuran. This feature could be related to the abnormally high DNA content of urodele cells. As in the DNA-DNA hybridization, RNA from X. Levis embryos hybridized less well with DNA from more distantly related species (Table 2).

(b) Melting behavior of hybridized DNA and RNA

If the hybridization of RNA or DNA fragments with DNA in agar implies hydrogen bonding between complementary bases, the melting point of the hybridized nucleic acids should be close to that of native DNA. The melting behavior of DNA-DNA

274 H. DENIS

and DNA-RNA duplexes was therefore examined. In one experiment, labeled DNA was incubated at 60°C with DNA in agar and the excess radioactive DNA was removed by rinsing in 2 x SSC at 60°C. The hybridized DNA was then eluted in 0.4 SSC by progressively raising the temperature, as shown in Fig. 2. Some of the entrapped DNA was found to leach out of the agar even at 42’C in O-4 SSC (Fig. 2).

TABLET

Hybridization of X. laevis RNA? with DNA from other species

Species o/o hybridization o/o of homologous Cross-reaction in the DNA-DNA

system$

Xenop bevis 9.0 100 100 Ranu ptpkns 2.3 26 28 Salmon 1.5 17 11 OX 1.6 18 6 Hen 0.3 3 2 Escherichia co.% 0 0 0

t 27pg of pulse-labeled RNA (4200 &/mm) from tail-bud embryos were incubated with 100 pg of DNA-agar.

$ Data taken from Table 1.

80

20

0 40 50 60 70 80 90

Temperature ("C)

FIG. 2. Melting behavior of DNA fragments hybridized with DNA in agar (-n--c]-).

17 pg of sonioated DNA (12,000 cts/min) were incubated with 190 pg of DNA in agar at 60°C. The unbound material was removed by rinsing with 100 ml. of 2 x SSC at 60°C. The agar was then transferred to a water-jacketed column, the salt concentration was lowered to 0.4 SSC and the temperature progressively raised from 40 to 85°C. Two lo-ml. fractions were collected at each temperature after 15 mm of equilibration. The amount of radioactive material released at each temperature was then determined. Total number of counts &ted: 1600 (1.3%). The optical melting curve of high molecular weight (-O-O-) and sonicated (-a-@-) DNA in 0.4 SSC is shown for comparison. The leaching of radioactive DNA trapped in agar and subjected to the same elution procedure as described above is also recorded (-I-a-). The figures shown next to the melting curves are the T, values of the DNA’s studied.


The amount of DNA lost remains rather small below 80°C. Above this temperature, the agar begins to dissolve and loses all the entrapped DNA. If allowance is made for the leaching of DNA from the agar, the DNA fragments are found to “melt” 17°C lower than native DNA. Apparently, the molecular structures formed between DNA fragments and DNA-agar are much less stable than those existing in native DNA. A similar observation was made by Walker & MacLaren (1965) in their study of mouse DNA.

A similar study was carried out on the stability of the RNA-DNA hybrids formed at different temperatures (Fig. 3). The hybrids formed at higher temperatures have higher melting points. At the same time, their melting curves become steeper. The T, of the hybrid formed at 72°C is only 5°C lower than the T, of native DNA. If allowance is made for the leaching of DNA from the ags,r during the elution procedure, the melting point of the 72°C hybrid is virtudly the same as that of DNA.

Temperature (“C)

Fra. 3. Melting behavior of DNA-RNA hybrids formed by incubation at different temperatures (37, 50, 60 and 72’C)

About 140 pg of r3aP]RNA (15,500 cts/min) from stage 28 embryos were incubated with 100 mg of agar containing 125 pg of DNA and washed at the temperature of incubation with 100 ml. of 2 x SSC. Elution procedure as in Fig. 2. The melting pro% of high molecular weight DNA in O-4 x SSC is shown for comparison (--A-A-). The horizontal figures shown next to the melting curves are the T’, values of the hybrids.

The increased thermal stability of the DNA-RNA hybrids formed at higher temperatures could be due to the involvement in the duplexes of stretches of DNA and RNA containing increasing proportions of G+C. The duplexes with the lowest T, might contain fewer G-C pairs and thus be eluted more easily. However, the base composition of the hybrids formed at different temperatures did not differ significantly.

276 H. DENIS

(c) DNA sequences involved in. hybrid formation at different temperatures

The experiments reported so far suggest that the degree of similarity required for duplex formation between DNA and RNA is less stringent as the temperature of incubation is lowered. Some regions of the DNA which are partially homologous with the RNA may be involved in hybrid formation at 37°C but excluded at higher temperatures. This supposition is confirmed by the experiment depicted in Table 3. Labeled RNA from embryos was incubated with DNA-agar at 72°C for 15 hours. The temperature of incubation was then changed as shown in Table 3. It is clear that pre-incubation at 72°C does not prevent a large amount of binding during a second incubation at 50°C or 37°C. Apparently, large regions of the DNA or of the RNA, which could not hybridize at 72”C, because they were too different, do so at lower temperatures.

TABLE 3

Amount of RNA hybridized with DNA-agar after a second incubation at different temperaturesj-

Temperature Cts/min hybridized of incubation after the second

(“(2 incubation

37 1012 60 552 00 377 72 373

t Five s8mples (00 pg; 11,000 ote/min) of [l*C]RNA from stage 28 embryos were incub8ted for 16 hr 8t 72°C with 100 mg of 8g8r containing 126 pg of DNA. One sample w8a waehed 8t 72°C and found to have hybridized 240 cts/min with DNA. Three other samples were removed end incubated one more day at 37.60 8nd 60°C. respectively. The fifth sample wee kept throughout at 72°C. All samples were weshed at the temperature of the second incubation, 8nd the amount of hybridized RNA was determined as ususl (last column).

(d) Percentage of DNA bound as a function of the amount of DNA offered for hybridization

If more and more DNA in agar is offered for hybridization to a small amount of DNA fragments, the percentage of binding increases (Fig. 4). A large excess of DNA in agar (700 to 1) is necessary to retain 50% of the input DNA fragments. The shape of the hybridization curve suggests that some DNA fragments (those binding at the lowest DNA/DNA ratio) f?nd a binding site more easily than others. The wide range of hybridization capacities of the DNA fragments cannot be due to differences in their molecular weight, since the DNA fragments which are able to hybridize with DNA-agar were found to have the same sedimentation coe&ient (about 4 to 10 s) in a sucrose gradient as the fragments which do not bind. Furthermore, the fragments which hybridize easily during a first incubation do so again if they are re-incubated with the same amount of DNA-agar. Similarly, the fragments which do not bind during a first incubation show a low hybridization level in a second run. The same observation was made by Walker & MacLaren (1966) on mouse DNA.


I I IO 100

DNA in agar/DNA fragments

IO

FIQ. 4. Hybridization of DNA fragments with increasing amounts of DNA in agar.

6 pg of [i*C]DNA (3600 cts/min) were incubated 8t 60°C with increasing 8mOmts of agar containing erythrocyte DNA. Volume of incubation mixture: 1 ml. of 2 x SSC/g of agar.

(e) Hybridization of labeled embryonic RNA with increasing amounts of DNA-ugar

When pulse-labeled RNA from mouse liver is hybridized with increasing amounts of DNA in agar, the percentage of radioactive material bound increases according to a complex curve which presents an initial steep portion, followed by a relatively flat part (Hoyer et al., 1963). RNA from embryos exposed to l*CO, for one hour behaves in a similar way (Pig. 5 (b) and (c)) as well as RNA labeled with 32P from the beginning of development (Pig. 6 (a)). The shape of the curves shown in Figs 6 and 6 indicates that labeled RNA from embryos of different stages is formed of molecules with a wide range of hybridization capacities. There are molecules which hybridize at a low DNA/RNA ratio, i.e. which bind even when little DNA is available. Other molecules do not hybridize unless a large excess of DNA is present. The same behaviour can be observed when DNA fragments are hybridized with DNA in agar (Pig. 4).

Stage-36to38

4 (a) Stage 0 * A, , ; , , , , : I I 2 4 6 8 IO 12 14 16 I8 20 22

DNA/RNA

FIU. 5. Hybridization of pulse-labeled RNA from gastrula (stage 11, (b)-A-A-), tail-bud embryo (stages 24 to 26, -@-a--) and s wimming tadpole (st8ges 36 to 38, -O-O-), with increasing amounts of DNA-agar. The figures shown on the ordinate refer to the percentage of input radioaotive material remaining bound to DNA-8g8r at the end of the incubation. The hybridization of RNA from unfertilized eggs ((a)-A-A-) labeled several months before with saP is also recorded.

278 H. DENIS

0 4 8 12 16 20 24 28 32 DNA/RNA

5 ‘3 ,o IO

s _ ‘C D r” 8 x -

6-

4-

2-

(b)- I I 1 I I I

0 20 40 60 80 100 120 Development (hr)

FIQ. 6. Hybridization of RNA from embryos exposed to azP from the beginning of development (neurule, (stages 17 to 20, -O-O-), tail-bud embryo (stages 22 to 24, -O-e--) and swimming tadpole (stages 33 to 36, -I-J--~-; stage 42, -B-H--)) with inore&ng amounts of DNA-agar (a). The values plotted in (b) are taken from the hybridization curves of (a) and from five others not shown here, at the point where these curves become linear.

There is no detectable break in the hybridization curve of RNA from mature eggs labeled with 32P during oogenesis (Fig. 5 (a)).

As already shown by Hoyer et al. (1963) for mouse liver RNA, the distinction between hybridizable and non-hybridizable RNA appears again if a second cycle of incubation is carried out (Fig. 7). Pulse-labeled RNA which hybridizes in a first incubation binds again in the second run. In contrast, very little of the RNA which fails to hybridize in the first incubation binds in the second cycle, when it is incubated with an additional portion of fresh DNA.

(f) Base composition of hybridizable RNA synthesized by developing embryos

The base composition of the hybridizable and non-hybridizable [3aP]RNA fractions is shown in Table 4. The G+C content of non-hybridizable RNA is close to that of sRNA and rRNA (60% G+C) an increases slightly during development. In d embryos exposed to 3aP from fertilization onwards, an increasing proportion of the labeled RNA is represented by sRNA and rRNA, which accumulate during development. The base composition of bulk RNA thereby becomes closer to that of sRNA and rRNA (Brown & Littne, 1964). Hybridizable [3aP]RNA from gastrulae and neurulae has the same composition as DNA (42% G+C; Dawid, 1965). Hybridizable RNA from older embryos has a higher G+C content.

GENE EXPRESSION IN AMPHIBIAN DEVELOPMENT

80

70

60

50

40

30

20

IO

0 5 IO 15 5 IO 15 5 IO 15

Fraction no.

279

FIU. 7. Re-incubation of bound and unbound [l*C]RNA with DNA-agar. 1200 pg of pulse- labeled RNA from swimmin g tadpoles were inoubated with 1 g of agar containing 420 pg of RNA. The elution pattern of this RNA with 2 x SSC at 60°C (fractions 1 to 10) and with 0.01 SSC at 75°C (fraotions 10 to 15) is shown on left. Half of each peak was then re-incubated with 0.5 g of the same DNA-agar. Upon re-incubation, the front peak gave the elution pattern shown in the middle. The back peak produced the elution pattern shown on the right.

TABLET

Base composition of [32P]RNAt from embryos of different stages

Stage Non-hybridized RNA Hybridized RNA

Molar ratio % G+c Molar ratio % G+c

12-14 C 23 67 c 18 42 (fs&rW G 34 G 24

A 26 A 26 U 18 u 32

16-19 C 29 66 C 19 40 (neurula) G 26 G 21

A 26 A 33 u 20 U 27

26-28 c 27 68 c 24 47 (tail-bud) G 31 G 23

A 24 A 27 U 18 U 26

38-40 C 31 69 C 26 49 (swimming G 28 G 23 tadpole) A 23 A 27

U 18 U 24

42 C 31 61 (swimming a 30 tadpole) A 22

U 17

DNA (encording to Dawid, 1966)

C 21 42 G 21 A 29 T 29

t 110 H of RNA incubated with 250 mg of agar containing 220 pg of DNA. The embryos were exposed to aaP from the beginning of development.

19

280 H. DENIS

(g) Stability of mRNA in developing embryos

The hybridization level of [3aP]RNA decreases during development (Fig. 6 (b)). The decreasing proportion of radioactivity present in the hybridizable fraction suggests that this fraction turns over more rapidly than the non-hybridizable fraction.

The stability of mRNA in early embryos has been examined in a pulse-chase experiment. A large batch (1500) of gastrulae was labeled with 14C02 for one hour, washed free of isotope and allowed to develop for 60 hr. At intervals, groups of 100 embryos were removed and extracted. As shown in Fig. 8, the embryos synthesized labeled RNA in the first 40 hours of chase, which means that during all that time, a pool of radioactive precursors was present. The chase is assumed to be complete

5oc

2. - 4oc z” d 5 3oc

g 2 al 2oc .c E

-2 i; IOC

C I I I I I

IO 20 30 40 50 12 19 20 27 37-38 41 Stages

1 Hours of chase (22°C)

FIU. 8. Pulse-chase experiment performed with stage 12 (g&x&) embryos. 1600 embryos were labeled for 1 hr in W02 and then kept in non-radioactive medium for 60 hr. At the end of the chase, the embryos had reached stage 41. At the times shown, groups of 100 embryos were removed and extracted. -e--e-, Total radioactivity in RNA; -O--O-, cte/min hybridized after incubating 180 pg of RNA with 90 pg of DNA in agar. Both sets of values given in cts/min per embryo.

by 40 hours, since the total radioactivity in RNA does not change later on. The amount of radioactive material in hybridizable RNA increases rapidly during the first 10 hours of chase and then drops steeply (Fig. 8). The same experiment performed with stage 42 embryos is described in Fig. 9. Here again the pool of nucleic acid precursors remains radioactive long after (35 hours) the end of the pulse. In both experiments, the percentage of radioactive RNA that can be hybridized with DNA decreases steadily during the chase, so that at the end of the experiment the non- hybridized RNA forms 99% of the labeled RNA. This again shows that the hybridized RNA turns over more rapidly than the non-hybridized RNA, which accumulates during development.

The low efficiency of the chase after a 14C0, pulse makes it impossible to determine the half-life of hybridizable RNA. In both experiments the label present in the hybridized fraction decays with a half-period of about 20 hours (Pigs 8 and 9). As long as radioactivity is present in the precursor pool, the real turnover rate of hybridizable RNA must be faster than the observed turnover of the label in that fraction. Therefore the average half-life of hybridized RNA is certainly less than 20 hours, with no lower limit assignable.


0 I I I I IO 20 30 40 50

Hours of chase (22’ C)

FIG 9. Same experiment aa iu Fig. 8, performed with swimming tadpoles (stage 42). -a-@--, Total radioactivity in RNA; -O--O-, cts/min hybridized after incubating 50 pg of RNA with 70 pg of DNA in &g&r. Both sets of values are given in cts/min/embryo.

4. Discussion The influence of the incubation temperature on the stability of the DNA-RNA

hybrids (Fig. 2) suggests that the nucleotide sequences present in RNA can hybridize with a variety of DNA stretches bearing a more or less complete resemblance to themselves. Such a redundancy or near redundancy probably exists in the mouse genome (Britten, 1964) and could account for the low stability of the duplexes formed between RNA and DNA and between DNA fragments and DNA in agar (Walker $ McLaren, 1965). This property, if present also in the amphibian genome, could explain why some DNA fragments hybridize with DNA in agar more readily than others (Fig. 4). The DNA fragments which do not bind even at the highest DNA/DNA ratio might represent nucleotide sequences of very rare occurrence in the X. Levis genome. The complexity of the vertebrate genome prevents any significant re- naturation of single-stranded DNA in free solution (Marmur & Doty, 1961). In the DNA-agar system only the most redundant sequences would renature readily with fragmented DNA. A large portion of the entrapped DNA, however, can be hybridized if the concentration of added DNA fragments is sufficiently high to provide multiple copies of even the rarest sequences (Hoyer et al., 1964).

The accuracy with which the nucleotide sequences are paired in the DNA-DNA and DNA-RNA duplexes depends upon the hybridization conditions. In that respect, the situation found in bacteria is different (Bolton & MacCarthy, 1964), since the structures formed between DNA in agar and DNA or RNA have a melting point which is very close to that of native DNA. In X. laevis, the species-dependence of the hybridization between DNA in agar and DNA or RNA (Tables 1 and 2) shows that a considerable homology is required between the nucleic acids involved, in order for hybridization to occur. In spite of this, some mismatched sequences are included in the duplexes, especially in those formed at low temperatures (37°C and 50°C). In these “hybrids”, many nucleotide sequences of RNA are likely to be associated with

282 H. DENIS

regions of DNA different from those on which they have been transcribed (Table 3). Such errors must be excluded in the hybrids formed at 72”C, since the melting behaviour of these is almost identical to that of native DNA.

The presence of mismatched sequences in the hybrids resulting from an incubation at 60°C somewhat reduces the validity of the hybridization technique as a means of comparing several mRNA populations. Two mRNA molecules transcribed on partially redundant, i.e., similar but not identical DNA stretches, will have the same hybridization characteristics. Therefore they will not be distinguishable. Furthermore, the RNA which is readily hybridizable with DNA does not necessarily form a complete and uniform sample of a given mRNA population. The proportion of mRNA that hybridizes with DNA depends both on the DNA/RNA ratio in the incubation mixture and on the number of sites with which each mRNA molecule can pair in the DNA. This number of sites may vary from one to one hundred thousand (B&ten, personal communication). These considerations make it difficult to interpret the data of Fig. 5. The RNA molecules that hybridize readily with DNA, i.e., in the non-linear part of the hybridization curves, are likely to be mRNA. The RNA molecules which bind only at a high DNA/RNA ratio can be both (a) molecules represented in the genome in a small number of complementary sites and (b) molecules present in the cell in multiple copies per DNA site. sRNA and rRNA are molecules of the latter type, since they are complementary to small regions of the DNA (Yankofsky & Spiegelman, 1962; Ritossa & Spiegelman, 1965; Attardi, Huang & Kabat, 1965; Goodman & Rich, 1962; Giacomoni & Spiegelman, 1962; MacFarlane & Fraser, 1964). Therefore the RNA binding at a high DNA input oertainly oontains, besides mRNA, some sRNA and rRNA.

The presence of sRNA and rRNA in the hybridized fractions can explain the increase in the G+C content of these fractions during development (Table 4). Little newly synthesized and consequently labeled sRNA and rRNA is present in early embryos (Brown & Littna, 1964). Therefore the contribution of these types of RNA to the hybridized fractions is small and does not influence the base composition of these fractions. The situation changes in later development. 3aP-labeled sRNA and rRNA become increasingly radioactive and contribute an increasing proportion of G+C-rich RNA to the hybridized fractions.

In conclusion, the existence of a considerable amount of redundancy in the animal genome complicates the interpretation of the hybridization experiments. To allow a qualitative and quantitative comparison between the mRNA’s from embryos of different stages, it will be assumed first that mRNA from one given stage can be hybridized only with the DNA stretches on which it has been transcribed, and second that all the mRNA present in a given RNA preparation becomes bound to DNA when enough DNA is offered for hybridization, i.e., at the DNA/RNA ratio where the hybridization curves (Figs 5 and 6) become linear. These assumptions certainly over-simplify the situation. Their influence on the significance of the results will be dis- cussed in the accompanying paper. The properties of hybridizable RNA from embryos were found not to change significantly from one stage to another. Throughout development, 12 to 14% of the RNA synthesized during one hour hybridizes readily with DNA. The average half-life of hybridizable RNA is less than 20 hours both in early embryos and in differentiated tadpoles. The base composition of hybridized RNA increases slightly during development. This increase, however, may not reflect a real change in the chemical composition of mRNA itself.


This work w8a carried out while I w8a 8 qualified investigator of the Fonda National de la Recherche Scientifique.

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dam: North Holland Publ. Co. Ritossa, E. M. & Spiegelman, S. (1966). Proc. Nat. Acud. Sci., Wash. 53, 737. Scherrer, K., Lethem, H. BE Darncll, J. E. (1963). Proc. Nat. Ad. Sci., W’aah. 49, 240. Townes, P. L. (1953). Ezp. Cell Res. 4, 96. Walker, P. M. B. t MacL8ren, A. (1965). J. Mol. BioZ. 12, 394. Yankofsky, S. A. & Spiegelman, S. (1962). Proc. Nat. Acud. Sci., Wash. 48, 1069.

gene expression in amphibian development

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