Characterization of Yolk DNA from Xenopus Laevis Oocytes Ovulated in Vitro (Toad/Cytochemistry/Biochemistry/Yolk Platelets/Ambystoma)

Proc. Nat. Acad. Sci. USA Vol. 69, No. 5, pp. 1322-1326, May 1972

Characterization of Yolk DNA from Xenopus laevis Oocytes Ovulated In Vitro (toad/cytochemistry/biochemistry/yolk platelets/Ambystoma)

F. HANOCQ, M. KIRSCH-VOLDERS, J. HANOCQ-QUERTIER, E. BALTUS, AND G. STEINERT Laboratoire de Cytologie et Embryologie Moleculaires, Universit6 Libre de Bruxelles, Brussels, Belgium Communicated by J. Brachet, February 28, 1972

ABSTRACT High molecular weight DNA was isolated cells, and blocked in metaphase of the second maturation from the yolk platelets of Xenopus laevis oocytes ovulated division. in vitro. Yolk DNA has the same buoyant density in CsCl gradients as mitochondrial and nuclear DNAs, and, like Yolk Platelets them, it is double-stranded. Yolk DNA behaves like nuclear were prepared by the method of Wallace and DNA, and not like mitochondrial DNA, upon renaturation Karasaki (3). The ovulated oocytes were homogenized in after denaturation. The molecules are always linear. 0.25 M sucrose-5% polyvinylpyrrolidone (ViPyr). (pH 7.0); Cytochemical and biochemical controls preclude the pos- 10 ml of medium were used for each 1000 oocytes. The ho- sibility that the yolk DNA might be contaminated by nu- mogenate was layered on an equal volume of 1 M sucrose- clear or mitochondrial DNA. We conclude that the yolk 5% DNA is an endogenous component of the yolk platelets. (ViPyr). and centrifuged for 20 min at 500 X g. The pellet was suspended in 0.25 M sucrose-5% (ViPyr). and centrifuged again in the same way. Electron microscopic controls showed As described in a previous paper (1), DNA could be extracted that contamination of the yolk platelets by pigment and from the yolk platelets isolated from whole ovaries of Xenopus mitochondria was negligible. laevis. Since it was likely that the yolk fraction was contaminated with nuclear DNA from the follicle cells in these earlier Mitochondria were isolated by Dawid's method (4). experiments, we now worked with oocytes that have been freed from their follicle cells in two manual removal Removal of the Jelly of Unfertilized Shed Eggs. For Xenopus, differentways: (i) a cysteine-papain mixture at pH 7.8 was used (4). of follicle cells after a short treatment of the with For Am- oocytes bystoma, the jelly was removed with forceps without any chemical agents, such as Pronase (0.15%) or sodium dodecyl vitro ovulation of the in chemical treatment. Both types of eggs were maintained in sulfate (0.3%); (ii) in oocytes the 0.1 Holtfreter's medium presence of hormones. In both cases, high molecular weight X (5). DNA could be extracted from the yolk platelets; the physico- DNA Determination. Instead of the fluorometric method chemical properties of yolk DNA from oocytes ovulated in (6), we used a modification of Keck's indol method (7): 75 vitro will be described. Our experimental data exclude beyond oocytes were homogenized in 2 ml of 0.25 M sucrose-0.03 M reasonable doubt the possible contamination of yolk DNA Tris HCl (pH 7.4)-0.1 M EDTA; 2 ml of cold 10% Cls- by either nuclear or mitochondrial DNA arising from follic- CCOOH were added; after 15 min, the pellet was centrifuged ular or other ovarian contaminating cells. We shall discuss and washed with cold 5% Cl3CCOOH. Lipids were removed the extraction of yolk DNA from unfertilized eggs of Xenopus by single successive washings with 94% alcohol at 40, 94% and Ambystoma mexicanum. alcohol at 500, n-butyl alcohol at 500, alcohol-ether (3: 1) at The was MATERIALS AND METHODS 500, and ether. pellet extracted for 20 min at 1000 with 600 ,l of 5% Cl3CCOOH and centrifuged. The DNA In Vitro Ovulation. Ovaries were removed from adult Xeno- content was estimated (6) on 250 Ml of the supernatant. For pus laevis, and the membranes of the ovarian follicles were estimation of the DNA content of the yolk platelets, 150 torn apart. Ovaries were then placed in amphibian Ringer's oocytes were used instead of 75. Tests for the specificity of solution containing, per liter, 10 mg of progesterone and an the method will be presented in Results. homogenate of four Xenopus pituitary glands. These two hormonal treatments are known to be most efficient in induc- Extraction of DNA from the Yolk Platelets. Yolk platelets ing, respectively, maturation and ovulation (2). After 5 hr, from ovulated oocytes were suspended in the following buffer the ovaries were transferred into plain sterile Ringer's solu- (10 ml for the yolk platelets of 1000 oocytes): 0.1 M glycine tion and left overnight in a sterile room at 200 in large open buffer (pH 10.6), 0.1 M EDTA, 1% NaCl, 6% sodium amino- petri dishes. The oocytes that had fallen off the ovaries and salicylate, 1% Triton X-100. This alkaline buffer dissolves that showed a white maturation spot were collected and the yolk platelets at once, without denaturing the DNA. The washed five times in Ringer's solution. Microscopic controls same volume of the following mixture was added: 11% with the Feulgen reaction showed them to be free of follicle freshly distilled cresol-O.07% 8-hydroxyquinoline-85% phenol (Mallinkrodt) (8). After the mixture was shaken for 30 min at room temperature, it was centrifuged for 10 min Abbreviations: (Vi Pyr)., polyvinylpyrrolidone; EthBr, ethidium at 12,000 X g. The supernatant was precipitated by the addi- bromide. tion of two volumes of ice-cold alcohol. After several hours at 1322 Downloaded by guest on September 27, 2021 Proc. Nat. Acad. Sci. USA 69 (1972) Yolk DNA in Amphibian Eggs 1323

40, the pellet was collected and dissolved in 0.15 M NaCl- were taken at 265 nm, and scanned with a Joyce-Loeb micro- 0.015 M Na3 citrate (1 X SSC). The solution was then centri- densitometer. The buoyant density was calculated by the fuged at 150 for 48 hr at 38,000 rpm in a Spinco SW50 rotor, method of Schildkraut et al. (11). after the addition of CsCl to an average density of 1.55, in Denaturation and Renaturation of DNA. 1-3 ,ug of DNA the presence of of ethidium bromide (EthBr) (9). 150,gg/ml were in of 0.1 SSC. Thermal denaturation: The fluorescent band of yolk DNA was collected, and the dissolved 750,ul X the solutions were heated for 15 min at 100°; they were then same centrifugation was repeated on a fresh EthBr-CsCl gradient. EthBr was subsequently removed by three succes- cooled immediately to -5°. Thermal renaturation: the solu- sive extractions with isoamyl alcohol (10), and the solution tions of denatured DNA were made up in 4 X SSC immediately after heating and left overnight at 600. containing DNA was dialyzed for 5 hr against 0.1 or 1 X SSC. Phage DNA was treated in the same way and used as a Mitochondrial DNA was obtained by the same method as control for the validity of the methods. The various prepara- yolk DNA. tions were studied by analytical centrifugation. Extraction of Nuclear DNA. Blood was collected from male Examination Under the Electron Microscope. Spreading of Xenopus in 2 X SSC, and DNA was extracted from the the DNA was performed by the method of Kleinschmidt and erythrocytes by the same method as for yolk DNA. Zahn (12). The hypophase was 0.1 M ammonium acetate- 0.5% formaldehyde. The DNA sample contained 2.5% Analytical Ultracentrifugation. The solutions containing formaldehyde. The electron micrographs were examined DNA were dialyzed against 0.1 X SSC. Suprapur CsCl was under an AEI model EM 6 B electron microscope after circu- added to a density of 1.700. Micrococcus lysodeikticus (luteus) lar shading with uranium oxide. DNA (p = 1.731 g/cm3) was used as a reference. The centrifugation was performed at 44,770 rpm for 20 hr at 200 in a Staining of Histological Sections with [3H]Actinomycin. Spinco model E analytical ultracentrifuge. Photographs Ovarian and ovulated oocytes were fixed by freeze substitu- tion. lO-um Sections were dipped into a [3H]actinomycin TABLE 1. Fluorometric and indole colorimetric determination solution for 60 min (20 ,Ci/ml; 8.4 Ci/mmol), washed for of DNA in whole ovulated Xenopus oocytes, in unfertilized 60 min in unlabeled actinomycin (3 ug/ml), and then washed eggs, and in their yolk platelets overnight under running tap water. The specificity of the actinomycin-binding test was established by treatment of A. Fluorometric determinations some of the slides with DNase (0.1 mg/ml) for 90 min at 370 (DABA)* in 0.01 M Tris-HCl (pH 7.4)-i mM MgSO4. K2 Ilford emul- (gg DNA/egg) sion was applied to the cytological preparations, and auto- % of radiographs were developed after 8 days of exposure. Whole Yolk total Preparation of Radioactive Chromatin. Xenopus kidney cells homogenate platelets egg were cultured for 3 days in HAMS F12 medium containing (a) Ovarian oocytes, 5 uCi/ml of [3H]thymidine (26 Ci/mmol). Thereafter, the (follicle cells re- cells were "chased" during 1 day in the same medium con- moved by sodium taining 4.6 u~g/ml of unlabeled thymidine. These radioactive dodecyl sulfate cells were homogenized with alumina in Ringer's solution. treatment) The low-speed pellet (undisrupted cells) was discarded, and Diameter the supernatant, containing radioactive chromatin fragments, 0.65 mm 0.011 was saved. 1.13mm 0.032 1.35 mm 0.038*t 0.020t 53t RESULTS (0.030-0.043) (0.016-0.024) (50-56) DNA Determinations. (i) Specificity of the reaction. Keck (7) B. Colorimetric determinations showed that the extent of interference with the DNA estima- (Indole) tion by several substances is low. We checked (data not shown) (gg DNA/egg) the influence of substances known to be highly concentrated Diameter 0.034t 0.017t 47.5t in the eggs: RNA, lipids, glycogen, and phosvitin. Only RNA 1.35 mm (0.33, 0.036) (0.015, 0.018) (45, 50) gives a slight interference: the excess color due to its presence (b) Oocytes after is 5% if the estimation is made on a mixture of RNA and DNA ovulation in vitro in the proportion of 100 to 1. This proportion is that found in 0.037§ 0.018§ 50§ the whole oocytes. No interference was found for phosvitin, (0.028-0.052) (0.015-0.023) (44-54) glycogen, and oocyte lipids. (c) Unfertilized shed (ii) DNA content of ovulated oocytes, of unfertilized eggs, and eggs of their yolk platelets. The results are expressed in Table 1. 0.035§ 0.014§ 40§ The same DNA values were obtained by the fluorometric (0.020-0.053) (0.008-0.018) (34-44) and colorimetric methods. The proportion of DNA contained in the yolk platelets is about 50% of the total egg DNA. Part A Taken from ref. 6. DABA = 3,5-diamino-benzoic acid. of the table is taken from a former publication (6), where it t Average of three determinations. was shown that 70-75% of the measured DNA could be t Average of two determinations. digested by the combined action of trypsin and DNase. The § Average of five determinations. fact that the individual estimations exhibit a great deal of Downloaded by guest on September 27, 2021 1324 Cell Biology: Hanocq et al. Proc. Nat. Acad. Sci. USA 69 (1972)

variation, for oocytes as well as for unfertilized eggs, is not DNA DNA DNA surprising in view of the highly variable sizes of eggs in differ- YOLK NUCL MIT ent batches (1.0-1.35 mm in diameter); the DNA content 31 1700 1731 seems to be proportional to the egg volume (6). The amount FIG.91.Analytica7 of DNA per egg is about 10,000 c (c, complement: amount of DNA per haploid set of chromosomes), in agreement with the 1099 estimations made by other authors (13). NAT Yield of Extraction of Yolk-DNA. The method used for the extraction of yolk DNA is, amongst all those we tried, the one that gives the cleanest preparations and the best yield. The usual chromatography on a methylated albumin- kieselguhr (MAK) column cannot be used for purification of 1 71 yolk DNA, since it is heavily contaminated by phosvitin, 1710 1716 which follows DNA in the aqueous phase of phenolic extracts. We have previously shown that yolk DNA is not retained by a MAK column unless it has been previously run on a CsCl gradient (14). The yield of extraction was established by Keck's method, by comparison of the amount of DNA present in the yolk pellet 1715 1706 with the amount of DNA recovered from the pellet after extraction; the yield is around 10%. Although it is rather low, it is not surprising that yolk DNA could not be purified with- RENAT out large losses, since 1 Mug of yolk DNA is diluted in several milligrams of proteins. The nature of the association between DNA and phospho- protein of the oocyte will be the subject of another publication. (Kirsch, M., in preparation). The strong binding between DNA and phosvitin could explain the low yield of yolk DNA FIG. 1. Analytical ultracentrifugation in CsCl of yolk, mito- upon extraction. chondrial (MIT), and nuclear (NUCL) DNAs in their native (NAT), denatured (DENAT), and renatured (RENAT) forms Studies on Thermal Denaturation and Renaturation by (Xenopus ovulated oocytes). Analytical Ultracentrifugation. Fig. 1 shows the densities of native, denatured, and renatured yolk DNA of Xenopus laevis, compared to those of nuclear DNA and mitochondrial DNA. Controls of Contamination of Yolk DNA by Nuclear DNA. One can see that yolk DNA has, in CsCl gradients, the same As shown by repeated microscopic controls, the process of density (1.699) as the nuclear and the mitochondrial DNAs in vitro ovulation of the oocytes completely removes all ad- studied by Dawid (15). Yolk DNA, as well as nuclear and hering follicle cells. However, one could still argue that these mitochondrial DNAs, denatures with a shift in its buoyant cells might partially disintegrate during the ovulation process: density, indicating that it is double-stranded. Like nuclear follicle cell chromatin might stick to the ovulated eggs, DNA, it retains about the density of the denatured form after without being detectable by the Feulgen reaction under the renaturation. Mitochondrial DNA, on the other hand, microscope; it might afterwards contaminate the yolk platelet acquires a density (1.706) when renatured that is intermediate preparation. This hypothesis was ruled out by two different between the native and denatured forms. This difference be- control experiments. tween yolk DNA and mitochondrial DNA demonstrates that (i) The very sensitive cytochemical method (17) of binding our preparations of yolk platelets were devoid of mitochon- to DNA of labeled actinomycin was used for detection of the drial contamination. eventual presence of chromatin fragments on the surface of the oocyte. Ovulated oocytes, as well as control ovarian Electron Microscopy on Yolk DNA and Nuclear DNA Mole- oocytes, were examined. All along the surface of the ovarian cules. Fibers of yolk DNA are always linear. No circular oocytes, black areas were observed on the nuclei of the follicle molecules were ever observed in our preparations of yolk cells. As expected, this labeling was no longer seen in the DNA. This result again precludes the possibility of con- DNase-treated sections. In contrast to the ovarian oocytes, tamination by mitochondrial DNA, since the latter consists no silver grains could be seen on the surface of the ovulated of circular molecules measuring about 5.4,m (16). 71 Mole- oocytes. In view of the sensitivity of the method used, one cules of nuclear DNA and 58 molecules of yolk DNA have can conclude that no detectable amount of exogenous been measured. The average size is about 20-24 ,um for nu- chromatin sticks to the surface of the ovulated eggs (Figs. 2 clear DNA and around 12-16 Mm for yolk DNA. We do not and 3). consider as necessarily significant the difference in sizes ob- (ii) The possible attachment of exogenous chromatin on served between the yolk and nuclear DNA molecules, since the surface of the ovulated oocytes has been studied in yolk DNA is much less concentrated in the extraction medium another way; since it is impossible to obtain labeled follicle than nuclear DNA. But, it is obvious that the DNA extracted cells, we have used chromatin from Xenopus kidney cells from the yolk platelets of Xenopus laevis oocytes is of high labeled in vitro with [3H]thymidine, as described in Methods. molecular weight. The chromatin suspension was added to the ovulated oocytes Downloaded by guest on September 27, 2021 Proc. Nat. Acad. Sci USA 69 (1972) Yolk DNA in Amphibian Eggs 1325

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FIG. 2. (Left) [3H]Actinomycin binding: section through two FIG. 3. (Right) ['H]Actinomycin binding: section through two ovarian oocytes; 3H-labeled follicle cell nuclei are visible outside oocytes ovulated in vitro. No radioactivity can be detected on the the pigmented layer of the oocytes. surface of the oocytes, either at the pigmented animal pole or at the vegetative one.

under the following conditions: samples of 100 oocytes were incubated with occasional shaking in 1.7 ml of Ringer's solu- or yolk platelets, tion, containing the labeled chromatin (1.7 ug DNA at 1.480 tected in the preparations of either whole eggs since no count above the uncontaminated controls was found in cpm/ug), for 10 min at room temperature. The amount (6) their respective extracts. Since we recover at least 0.1 ,ug of of DNA corresponded to 2800 follicular cells per oocyte. The purified DNA from the yolk platelets of 100 oocytes, if this oocytes were washed with three rinses of Ringer's solution. DNA was a nuclear exogenous contaminant, about 150 cpm Some of them were simply homogenized, and yolk platelets over the controls should have been recovered in the yolk were isolated from the others. The suspensions of whole eggs platelets of 100 oocytes. or of yolk platelets were precipitated by a final concentration of 5% Cl,3CCOOH at 40, washed at 40 with 5% Cl3CCOOH, DISCUSSION delipidated as described in Methods, and finally extracted DNA extracted from the yolk for Radioactivity The present work shows that twice with 5% Cl3CCOOH at 1000 20 min. platelets of ovulated eggs of Xenopus is of high molecular was in scintillation fluid of the 5% acid extracts then measured weight, linear, and double-stranded. Its density is 1.699, 1 g (PPO), 0.5 g [in toluene to liter, 5 of 2,5-diphenyloxazole like that of nuclear and of mitochondrial DNAs. 1 ,4-bis [2-(4-methyl-5-phenyloxazolyl) ]-benzene](POPOP)- of DNA by either nuclear or 2 of these Possible contamination yolk Triton X-100 (2:1 v/v). Table gives the results mitochondrial DNA has been ruled out by the following no could be de- measurements. It is clear that contamination evidence: (i) Repeated microscopic controls showed the absence of whole contaminating cells along the membrane of TABLE 2. Study of the possible contamination of ovulated the oocyte ovulated in vitro. On the other hand, a possible oocytes by radioactive Xenopus kidney cell chromatin (numbers contamination by chromosomal fragments, undetectable by correspond to duplicate batches extracted in the same way) the Feulgen reaction, has been excluded by two control experiments: (a) if labeled exogenous chromatin is added to Radioactivity, cpm ovulated oocytes, neither the whole-egg homogenate nor the Homogenate of 100 un- 36.8 i 7.5 yolk-platelets pellet exhibits any contaminant radioactivity contaminated oocytes 39.2 i 7.5 Average:38 and (b) if sections of ovulated oocytes are treated with ['H]- Yolk platelets isolated from 100 36.6 + 7.5 actinomycin, no accumulation of radioactive tracks can be uncontaminated oocytes 36.5 i= 7.5 Average: 37 detected on their surface. 1.7 ml of Ringer's solution (ii) A possible contamination of our preparations with containing kidney-cell mitochondrial DNA can also be excluded for the following labeled chromatin 2.848 + 215 Average: 2519 reasons: pellets of yolk platelets contain very few mitochon- (1 ,g of DNA/ml) dria, and preparations of yolk DNA contain no circular Homogenate of 100 oocytes in- molecules. Furthermore, during centrifugation in CsCl- cubated in 1.7 ml of the mitochondrial DNA should, because of its circular same Ringer's solution con- EthBr, taining kidney-cell labeled shape, be localized in a denser band than the yolk DNA. 38.4 Centrifugation of a mixture of the two types of DNA con- chromatin (1 1g of =1 7.5 DNA/ml) 37.5 i- 7.5 Average:Aeae338 firmed this surmise; yet no heavy band was observed when yolk Yolk platelets isolated from 100 DNA was centrifuged in CSCl-EthBr gradients. Finally, oocytes incubated in 1.7 ml renaturation experiments show that yolk DNA preparations of the same Ringer's solution never contain any rapidly renaturing component. containing kidney-cell There is, therefore, good reason to believe that DNA free labeled chromatin 38.0 i 7.5 from extraneous contamination can be isolated from the (1 Ag of DNA/ml) 34. 7 i 715 Average 36 yolk platelets of ovulated oocytes. The extraction and char- Downloaded by guest on September 27, 2021 1326 Cell Biology: Hanocq et al. Proc. Nat. Acad. Sci. USA 69 (1972)

acterization of the DNA present in the yolk platelets of We thank J. Verhulst for skillful assistance in performing the amphibian eggs confirms the findings of other workers, who analytical ultracentrifugations and Dr. P. Malpoix for kindly revising the English translation. We are very grateful to Dr. G. obtained indications for the presence of DNA in these particles Woolfe (Boots Pure Drug Co. Ltd, Nottingham) for a gift of (18, 19). Steinert and Van Gansen (19) showed by electron ethidium bromide. M.K-V. was supported by a fellowship of the microscopy that labeled actinomycin D binds to the yolk "Institut pour la Recherche Scientifique dans l'Industrie et platelets, especially in their outer layer. I'Agriculture." We recently isolated yolk DNA from unfertilized eggs of 1. Baltus, E., Hanocq-Quertier, J. & Brachet, J. (1968) Ambystoma mexicanum, and found that its buoyant density Proc. Nat. Acad. Sci. USA 61, 469-476. (1.699) is different from that of nuclear (1.704) and of mito- 2. Smith, L. D. & Ecker, R. E. (1971) Curr. Top. Develop. chondrial DNAs Yolk DNA Biol. 5, 1-38. (1.696) (20). is, thus, distinguish- 3. Wallace, R. A. & Karasaki, S. (1963) J. Cell Biol. 18, 153- able from the other two kinds of DNAs. However, extraction 166. of yolk DNA from unfertilized eggs of Ambystoma and 4. Dawid, I. B. (1965) J. Mol. Biol. 12, 581-599. Xenopus is not reproducible. A possible explanation for this 5. Holtfreter, J. (1963) J. Exp. Zool. 93, 251-323. variability might be that various metabolic changes take 6. Hanocq-Quertier, J., Baltus, E., Ficq, A. & Brachet, J. (1968) J. Embryol. Exp. Morphol. 19, 273-282. place in the egg when it leaves the ovary and passes through 7. Keck, K. (1956) Arch. Biochim. Biophys. 63, 446-457. the oviduct before spawning (21, 22). In particular, the oc- 8. Kirby, K. S. & Cook, E. A. (1967) Biochem. J. 104, 254- currence of a profound change has been shown in both the 257. internal distribution and content of Mg2+ and Ca2+ between 9. Radloff, R., Bauer, W. & Vinograd, J. (1967) Proc. Nat. Acad. the time of response of the hormonal Sci. USA 57, 1514-1521. oocyte to stimulation 10. Cozzarelli, N. R., Kelly, R. B. & Kornberg, A. (1968) and the moment that the egg is laid (22). This change in the Proc. Nat. Acad. Sci. USA 60, 992-999. concentration of divalent ions is concomitant with a massive 11. Schildkraut, C. L., Marmur, J. & Doty, P. (1962) J. Mol. disintegration of subcellular structures. In our own experi- Biol. 4, 430-443. ments (unpublished), simply leaving ovulated oocytes for a 12. Kleinschmidt, A. & Zahn, R. K. (1959) Z. Naturforsch. B. 14, 770-779. few minutes in Ringer's solution supplemented with 10-2 M 13. Williams, J. & Weber, R. (1965) in The Biochemistry of Mg2+ results in a considerable fall in the amount of DNA Animal Development (Academic Press. N.Y.), Vol. 1, pp. that can subsequently be extracted from the yolk platelets. 13-71. The extraction of DNA from the yolk platelets of unfertil- 14. Hanocq-Quertier, J., Baltus, E., Hanocq, F., Kirsch, M. & G. Arch. eggs thus be more Steinert, (1970) Int. Physiol. Biochim. 78, 998-1000. ized might made reproducible by modifi- 15. Dawid, I. B. & Wolstenholme, D. R. (1968) Biophys. J. cation of the ionic concentrations used. 8, 65-81. It is now necessary to reconsider the exact role of the yolk 16. Dawid, I. B. & Wolstenholme, D. R. (1967) J. Mol. Biol. platelets: it is known that they contain reserve substances 28, 233-245. that are used during the growth of the embryo. It seems likely 17. Brachet, J. & Ficq, A. (1965) Exp. Cell Res. 38, 153-159. 18. Fernandez de Recondo, M. (1968) Arch. Bioquim. Quim. that they might contribute in a less-passive way to develop- Farm. 14, 3-15. ment, since recent experiments (23) have shown that they 19. Steinert, G. & Van Gansen, P. (1971) Exp. Cell Res. 64, also contain metabolically active RNA. The present paper 355-365. shows that, in Ambystoma at least, the high molecular weight 20. Wolstenholme, D. R. & Dawid, I. B. (1968) J. Cell Biol. 39, 222-228. yolk DNA is different from nuclear and mitochondrial DNAs. 21. Smith, L. D. & Ecker, R. E. (1970) Develop. Biol. 22, This peculiarity should make possible experiments that 622-637. would lead to an understanding of the biological role of yolk 22. Morrill, C. A., Kostellow, A. B. & Murphy, J. B. (1971) DNA during embryonic development. Exp. Cell Res. 66, 289-298. 23. Kelley, R. O., Nakai, G. S. & Guganig, M. E. (1970) J. Cell Biol. 47, la-104a 103. Downloaded by guest on September 27, 2021