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Ribosomal RNA synthesis in pre- and postgastrula embryos of xenopus laevis
Cell Differentiation 1,209-213 ( 1972). © North-Holland Publishing Company
R I B O S O M A L R N A S Y N T H E S I S IN PRE- A N D P O S T G A S T R U L A E M B R Y O S O F XENOPUS L A E V I S Richard LANDESMAN Department of Zoology, Universityof Vermont, Burlington, Vermont 05401
Accepted1 May1972 The synthesis of ribosomal RNA in pre- and postgastrula embryos of Xenopus laevis was examined using 3H-CH3-methionine. No incorporation of label into ribosomal RNA is seen in pregastrula stages be it whole cell or ribosome extracts. These data support the observation that during early embryogenesis of Xenopus the synthesis of ribosomal RNA is under regulation.
The stage-specific pattern of ribosomal RNA (rRNA) biosynthesis observed during early embryogenesis in sea urchins and amphibians suggests that specific control mechanisms are operating. The biosynthesis of rRNA is not detected prior to the onset of gastrulation, whereas after that time it is easily measured (Brown et al., 1964; Gurdon, 1968; Gross, 1968; Landesman et al., 1968; Bachvarova et al., 1968; Knowland, 1970). That observation has led some investigators to propose the presence of a specific repressor for rRNA during the pregastrula stages (Shiokawa et al., 1967; Crippa, 1970). It has been reported recently, however, that rRNA is synthesized during pregastrula stages in the sea urchin, and Xenopus, at a rate similar to that calculated for postgastrula stages (Emerson et al., 1970, 1971) and suggested that its detection has been rendered most difficult because its accumulation (rRNA) is masked at the earlier stages by DNA-like RNA. The above results are based on two rather important assumptions. One is that all of the radioactivity eluting from a methylated albumin kieselguhr (MAK) column under the conditions of its use (Emerson et al., 1970, 1971) and with the bulk of the rRNA, is in fact newly synthesized rRNA. There is evidence that this may not be the case (Sueoka et al., 1962; Ellem et al., 1964). The second assumption is that the rate of synthesis of rRNA for Xenopus is the same on a per nucleus basis for both pre- and postgastrula embryos; the authors present no evidence for that. The experiment reported herein was designed to further examine the possibility that rRNA is synthesized in pregastrula amphibian embryos. Previous data (Brown et al., 1964; Shiokawa et al., 1967; Bachvarova et al., 1968;Gross, 1968; Gurdon,
210
rRNA synthesis in embryos of Xenopus
1968; Landesman et al., 1968; Knowland, 1970; Crippa, 1970) on the pattern of rRNA synthesis have been obtained with the usual labels for RNA, i.e., 32p., 3H. ' 14C-uridine, etc. In the experiments reported here 3H-CH3-methionine was used as the source of label. CH3-methionine has been shown to be a specific labeling compound for transfer RNA (tRNA) and rRNA; both of these species are methylated whereas messenger is not (Comb et al., 1965; Greenberg et al., t966; Zimmerman et al., 1967; Muramatsu et al., 1968; Landesman et al., 1969). Pre- and postgastrula Xenopus embryos were labeled with the methionine for 4 hr. (A longer labeling time was not feasible since some of the pregastrula embryos would then reach the stage at which rRNA synthesis is normally detected.) The viability of the embryos was checked and development during the incubation period proceeded normally. Whole-cell RNA was then extracted by standard methods; in addition ribosomes were isolated by high-speed centrifugation, and from them RNA (see figure legend for details). The latter procedure for isolating radioactive RNA associated with ribosomes is more specific than methods utilizing MAK column chromatography (Sueoka et al., 1962; Ellem et al., 1964). The RNA extracts were then analyzed on sucrose gradients and the results are shown in fig. 1. No incorporation into RNA sedimenting at 28S and 18S was seen in pregastrula material, either whole cell or ribosome extracts. It is clear, however, that rRNA is synthesized in postgastrula embryos. There are major radioactive peaks sedimenting at 28S and 18S as one would expect for rRNA. In addition, the high-speed postribosomal supernatant from each stage was examined for the distribution of label. The radioactivity associated with RNA from postgastrula stages was entirely coincident with optical density contributed by ribosomal subunit and 4S RNA, whereas the labeling pattern in the pregastrula preparation was not coincident with optical density except in the region of the 4S (not shown). The results also show that tRNA is labeled in both pxe- and postgastrula embryos, again as expected (Comb et al., 1965; Greenberg et al., 1966; Zimmerman et al., 1967; Muramatsu et al., 1968; Landesman et al., 1969). The comparatively low level of radioactivity associated with 4S RNA in extracts from pregastrula embryos as compared with postgastrula embryos has been observed before (Brown, 1964; Woodland et al., 1968; Knowland, 1970). A possible explanation is that there is a drastic alteration in the methionine precursor pool for RNA between the two developmental stages. On the basis of the 4S RNA incorporation data, one would expect some labeling of 28S and 18S RNA if those species were being synthesized. The absence of rRNA labeling in pregastrula embryos could occur if there was no methylation of the RNA as it was being processed; that is entirely contrary to our present understanding of rRNA biosynthesis and subsequent modification (Greenberg et al., 1966; Darnell, 1968; Loening et al., 1969). Pool data can be of little value since labeled amino acids can be incorporated directly into protein without equilibrating with the total free amino acid pool (Manchester, 1970). In Rana pipiens it has been demonstrated that even after an artificial increase in the leucine
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Fraction Number Fig. 1. Sucrose gradient centrifugation of RNA extracted from embryos and ribosomes of laevis. Following dissociation in Ca 2+- and Mg2+-frec Stearns' solution (Stearns et al., 1958), 150 embryos (pregastrula, stage 4; postgastrula, stage 17) per flask were incubated in complete Stearns' (2 ml) containing 100 #Ci/ml 3H-CH3-methionine (spec. act. 8 Ci/mmole, Amersham/Searle) for 4 hr at 20°C. Washed embryos were homogenized in 0.1 M acetate buffer (pH 5.0, 0.5% SDS). RNA was extracted with phenol-acetate and precipitated twice with 0.I M NaC! and 2 volumes of ethanol. Sedimentation was in a 13 ml linear sucrose gradient (15-30%, 0.1 M acetate pH 5, 0.5% SDS) at 24,000 rev/min for 17.5 hr (International B-60, SB-238 rotor) at 22°C. The gradients were eluted through a 2-ram flow cell of a Gilford spectrophotometer and an equal volume of 15% trichloroacetic acid (TCA) added to each fraction. Precipitable counts were trapped on Millipore filters and counted in diluted Spectrafluor (Amersham/ Searle). Counts (cts) due to background (25 cts/min) were subtracted from all .samples. Ribosome pellets were obtained from 150 embryos (of each stage) homogenized in 10 ml of 0.01 M Tris (pH 7.0, 0.25 M sucrose, 10-3 M MgC12, 5 #g]ml polyvinyl sulfate). Following a preliminary spin at 2000 g for 5 rain, sodium deoxycholate was added to the supernatant (final concentration 0.5%) and this mixture centrifuged at 10,000 g for 10 min. The clear supernatant was then centrifuged for 120 rain at 55,000 rpm (International B-60, A-321 rotor). The pellet was used for RNA extraction as outlined above. U p p e r l e J t - sedimentation pattern for RNA extracted from pregastrula embryos. U p p e r r i g h t - sedimentation pattern for RNA extracted from ribosome pellet. L o w e r l e f t - sedimentation pattern for RNA extracted from postgastrula embryos. L o w e r r i g h t - sedimentation pattern for RNA from ribosome pellet from postgastrula embryos. - optical density at 260 nm; o--cts/min. Xenopus
212
rRNA synthesis in embryos of Xenopus
pool its incorporation into protein remained unchanged (Ecker et at., 1968). In addition, amino-acid pool data derived from eggs and embryos of R. pipiens show that methionine remained at an extremely low level ( < 0.1/amole per 104 embryos) from fertilization to the 16-ceU stage (Ecker et al., 1968). The results reported here agree with other data that show that pregastrula rRNA synthesis is not detectable in sea urchin embryos using 14C-CH3-methionine (Comb et al., 1965), or in Xenopus using uridine labeling combined with electrophoretic analysis of RNA (Knowland, 1970; Abe et al., 1971). The possibility still remains that the combination of a change in precursor pool and the total number of incorporating nuclei per embryo (equal numbers of embryos were used in all the experiments reported herein) would render rRNA biosynthesis undetectable by the methods used. Calculations based on the measurement of the number of nuclei per embryo at the different stages have shown that the amplification factor (about 10×) is not sufficient to render rRNA biosynthesis undetectable (Gurdon et al., 1968). Data on pool sizes and changes from later embryonic stages are not presently available. Further experimentation is underway to analyze those problems and to examine the control mechanisms involved in the regulation of rRNA biosynthesis during development.
ACKNOWLEDGMENTS This investigation was supported by an NSF Institutional Grant provided for by the University of Vermont. I thank Dr. Robert Low for his critical comments.
REFERENCES Abe, H. and K. Yamana: Biochem.Biophys. Acta 213,392-406 (1971). Bachvarova, R., E.H. Davidson, V.G. Allfrey and A.E. Mirsky: Proc. Natl. Acad. Sci. US 55, 358-365 (1968). Brown, D.D.: J. Exptl. Zool. 157,101-114 (1964). Brown, D.D. and E. Littna: J. Mol. Biol. 8,669-687 (1964). Comb, D.G., S. Katz, R. Branda, C.J. Pinzino: J. Mol. Biol. 14,195-213 (1965). Crippa, M.: Nature 227, 1138-1140 (1970). DarneR, J.E.: Bacteriol. Rev. 32, 262-290 (1968). Ecker, R.E. and L.D. Smith: Develop. Biol. 18,232-249 (1968). Ellem, K.A.O. and J.W. Sheridan: Biochem. Biophys. Res. Commun. 16,505-510 (1964). Emerson;C.P. Jr. and T. Humphreys: Science 171,898-901 (1971). Emerson, C.P. Jr. and T. Humphreys: Develop. Biol. 23, 86-112 (1970). Greenberg, H. and S. Penman: J. Mol. Biol. 21,527-535 (1966). Gross, P.R.: Ann. Rev. Bioehem. 37,631-660 (1968). Gurdon, J.B.: EssaysBiochem. 4, 25-68 (1968). Gurdon, J.B. and H.R. Woodland: Biol. Rev. 43,233-267 (1968). Knowland, J.S.: Biochem. Biophys. Acta 204,416-429 (1970).
R. LANDESMAN
213
Landesman, R. and P.R. Gross: Develop. Biol. 18,571-589 (1968). Landesman, R. and P.R. Gross: Develop. Biol. 19, 244-260 (1969). Loening, U.E., K.W. Jones and M~L.Birnstiel: J. Mol. Biol. 45,353-366 (1969). Manchester, K.L.: Biochem. Actions Hormones 1,267-281 (1970). Muramatsu, M. and T. Fujisawa: Biochem. Biophys. Acta 157,476-492 (1968). Shiokawa, K. and K. Yamana: Develop. Biol. 16, 389-406 (1967). Stearns R.N. and A.B. KosteUov: in: The Chemical Basis of Development, eds. W.D. McElroy and B. Glass (Johns Hopkins Press) (1958). Sueoka, N. and T.Y. Cheng: J. Mol. Biol. 4,161-172 (1962). Woodland, H.R. and J.B. Gurdon: J. Embryol. Exptl. Morphol. 19, 363-385 (1968). Zimmerman, E.F. and B.W. Holler: J. Mol. Biol. 23, 149-161 (1967).
R I B O S O M A L R N A S Y N T H E S I S IN PRE- A N D P O S T G A S T R U L A E M B R Y O S O F XENOPUS L A E V I S Richard LANDESMAN Department of Zoology, Universityof Vermont, Burlington, Vermont 05401
Accepted1 May1972 The synthesis of ribosomal RNA in pre- and postgastrula embryos of Xenopus laevis was examined using 3H-CH3-methionine. No incorporation of label into ribosomal RNA is seen in pregastrula stages be it whole cell or ribosome extracts. These data support the observation that during early embryogenesis of Xenopus the synthesis of ribosomal RNA is under regulation.
The stage-specific pattern of ribosomal RNA (rRNA) biosynthesis observed during early embryogenesis in sea urchins and amphibians suggests that specific control mechanisms are operating. The biosynthesis of rRNA is not detected prior to the onset of gastrulation, whereas after that time it is easily measured (Brown et al., 1964; Gurdon, 1968; Gross, 1968; Landesman et al., 1968; Bachvarova et al., 1968; Knowland, 1970). That observation has led some investigators to propose the presence of a specific repressor for rRNA during the pregastrula stages (Shiokawa et al., 1967; Crippa, 1970). It has been reported recently, however, that rRNA is synthesized during pregastrula stages in the sea urchin, and Xenopus, at a rate similar to that calculated for postgastrula stages (Emerson et al., 1970, 1971) and suggested that its detection has been rendered most difficult because its accumulation (rRNA) is masked at the earlier stages by DNA-like RNA. The above results are based on two rather important assumptions. One is that all of the radioactivity eluting from a methylated albumin kieselguhr (MAK) column under the conditions of its use (Emerson et al., 1970, 1971) and with the bulk of the rRNA, is in fact newly synthesized rRNA. There is evidence that this may not be the case (Sueoka et al., 1962; Ellem et al., 1964). The second assumption is that the rate of synthesis of rRNA for Xenopus is the same on a per nucleus basis for both pre- and postgastrula embryos; the authors present no evidence for that. The experiment reported herein was designed to further examine the possibility that rRNA is synthesized in pregastrula amphibian embryos. Previous data (Brown et al., 1964; Shiokawa et al., 1967; Bachvarova et al., 1968;Gross, 1968; Gurdon,
210
rRNA synthesis in embryos of Xenopus
1968; Landesman et al., 1968; Knowland, 1970; Crippa, 1970) on the pattern of rRNA synthesis have been obtained with the usual labels for RNA, i.e., 32p., 3H. ' 14C-uridine, etc. In the experiments reported here 3H-CH3-methionine was used as the source of label. CH3-methionine has been shown to be a specific labeling compound for transfer RNA (tRNA) and rRNA; both of these species are methylated whereas messenger is not (Comb et al., 1965; Greenberg et al., t966; Zimmerman et al., 1967; Muramatsu et al., 1968; Landesman et al., 1969). Pre- and postgastrula Xenopus embryos were labeled with the methionine for 4 hr. (A longer labeling time was not feasible since some of the pregastrula embryos would then reach the stage at which rRNA synthesis is normally detected.) The viability of the embryos was checked and development during the incubation period proceeded normally. Whole-cell RNA was then extracted by standard methods; in addition ribosomes were isolated by high-speed centrifugation, and from them RNA (see figure legend for details). The latter procedure for isolating radioactive RNA associated with ribosomes is more specific than methods utilizing MAK column chromatography (Sueoka et al., 1962; Ellem et al., 1964). The RNA extracts were then analyzed on sucrose gradients and the results are shown in fig. 1. No incorporation into RNA sedimenting at 28S and 18S was seen in pregastrula material, either whole cell or ribosome extracts. It is clear, however, that rRNA is synthesized in postgastrula embryos. There are major radioactive peaks sedimenting at 28S and 18S as one would expect for rRNA. In addition, the high-speed postribosomal supernatant from each stage was examined for the distribution of label. The radioactivity associated with RNA from postgastrula stages was entirely coincident with optical density contributed by ribosomal subunit and 4S RNA, whereas the labeling pattern in the pregastrula preparation was not coincident with optical density except in the region of the 4S (not shown). The results also show that tRNA is labeled in both pxe- and postgastrula embryos, again as expected (Comb et al., 1965; Greenberg et al., 1966; Zimmerman et al., 1967; Muramatsu et al., 1968; Landesman et al., 1969). The comparatively low level of radioactivity associated with 4S RNA in extracts from pregastrula embryos as compared with postgastrula embryos has been observed before (Brown, 1964; Woodland et al., 1968; Knowland, 1970). A possible explanation is that there is a drastic alteration in the methionine precursor pool for RNA between the two developmental stages. On the basis of the 4S RNA incorporation data, one would expect some labeling of 28S and 18S RNA if those species were being synthesized. The absence of rRNA labeling in pregastrula embryos could occur if there was no methylation of the RNA as it was being processed; that is entirely contrary to our present understanding of rRNA biosynthesis and subsequent modification (Greenberg et al., 1966; Darnell, 1968; Loening et al., 1969). Pool data can be of little value since labeled amino acids can be incorporated directly into protein without equilibrating with the total free amino acid pool (Manchester, 1970). In Rana pipiens it has been demonstrated that even after an artificial increase in the leucine
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Fraction Number Fig. 1. Sucrose gradient centrifugation of RNA extracted from embryos and ribosomes of laevis. Following dissociation in Ca 2+- and Mg2+-frec Stearns' solution (Stearns et al., 1958), 150 embryos (pregastrula, stage 4; postgastrula, stage 17) per flask were incubated in complete Stearns' (2 ml) containing 100 #Ci/ml 3H-CH3-methionine (spec. act. 8 Ci/mmole, Amersham/Searle) for 4 hr at 20°C. Washed embryos were homogenized in 0.1 M acetate buffer (pH 5.0, 0.5% SDS). RNA was extracted with phenol-acetate and precipitated twice with 0.I M NaC! and 2 volumes of ethanol. Sedimentation was in a 13 ml linear sucrose gradient (15-30%, 0.1 M acetate pH 5, 0.5% SDS) at 24,000 rev/min for 17.5 hr (International B-60, SB-238 rotor) at 22°C. The gradients were eluted through a 2-ram flow cell of a Gilford spectrophotometer and an equal volume of 15% trichloroacetic acid (TCA) added to each fraction. Precipitable counts were trapped on Millipore filters and counted in diluted Spectrafluor (Amersham/ Searle). Counts (cts) due to background (25 cts/min) were subtracted from all .samples. Ribosome pellets were obtained from 150 embryos (of each stage) homogenized in 10 ml of 0.01 M Tris (pH 7.0, 0.25 M sucrose, 10-3 M MgC12, 5 #g]ml polyvinyl sulfate). Following a preliminary spin at 2000 g for 5 rain, sodium deoxycholate was added to the supernatant (final concentration 0.5%) and this mixture centrifuged at 10,000 g for 10 min. The clear supernatant was then centrifuged for 120 rain at 55,000 rpm (International B-60, A-321 rotor). The pellet was used for RNA extraction as outlined above. U p p e r l e J t - sedimentation pattern for RNA extracted from pregastrula embryos. U p p e r r i g h t - sedimentation pattern for RNA extracted from ribosome pellet. L o w e r l e f t - sedimentation pattern for RNA extracted from postgastrula embryos. L o w e r r i g h t - sedimentation pattern for RNA from ribosome pellet from postgastrula embryos. - optical density at 260 nm; o--cts/min. Xenopus
212
rRNA synthesis in embryos of Xenopus
pool its incorporation into protein remained unchanged (Ecker et at., 1968). In addition, amino-acid pool data derived from eggs and embryos of R. pipiens show that methionine remained at an extremely low level ( < 0.1/amole per 104 embryos) from fertilization to the 16-ceU stage (Ecker et al., 1968). The results reported here agree with other data that show that pregastrula rRNA synthesis is not detectable in sea urchin embryos using 14C-CH3-methionine (Comb et al., 1965), or in Xenopus using uridine labeling combined with electrophoretic analysis of RNA (Knowland, 1970; Abe et al., 1971). The possibility still remains that the combination of a change in precursor pool and the total number of incorporating nuclei per embryo (equal numbers of embryos were used in all the experiments reported herein) would render rRNA biosynthesis undetectable by the methods used. Calculations based on the measurement of the number of nuclei per embryo at the different stages have shown that the amplification factor (about 10×) is not sufficient to render rRNA biosynthesis undetectable (Gurdon et al., 1968). Data on pool sizes and changes from later embryonic stages are not presently available. Further experimentation is underway to analyze those problems and to examine the control mechanisms involved in the regulation of rRNA biosynthesis during development.
ACKNOWLEDGMENTS This investigation was supported by an NSF Institutional Grant provided for by the University of Vermont. I thank Dr. Robert Low for his critical comments.
REFERENCES Abe, H. and K. Yamana: Biochem.Biophys. Acta 213,392-406 (1971). Bachvarova, R., E.H. Davidson, V.G. Allfrey and A.E. Mirsky: Proc. Natl. Acad. Sci. US 55, 358-365 (1968). Brown, D.D.: J. Exptl. Zool. 157,101-114 (1964). Brown, D.D. and E. Littna: J. Mol. Biol. 8,669-687 (1964). Comb, D.G., S. Katz, R. Branda, C.J. Pinzino: J. Mol. Biol. 14,195-213 (1965). Crippa, M.: Nature 227, 1138-1140 (1970). DarneR, J.E.: Bacteriol. Rev. 32, 262-290 (1968). Ecker, R.E. and L.D. Smith: Develop. Biol. 18,232-249 (1968). Ellem, K.A.O. and J.W. Sheridan: Biochem. Biophys. Res. Commun. 16,505-510 (1964). Emerson;C.P. Jr. and T. Humphreys: Science 171,898-901 (1971). Emerson, C.P. Jr. and T. Humphreys: Develop. Biol. 23, 86-112 (1970). Greenberg, H. and S. Penman: J. Mol. Biol. 21,527-535 (1966). Gross, P.R.: Ann. Rev. Bioehem. 37,631-660 (1968). Gurdon, J.B.: EssaysBiochem. 4, 25-68 (1968). Gurdon, J.B. and H.R. Woodland: Biol. Rev. 43,233-267 (1968). Knowland, J.S.: Biochem. Biophys. Acta 204,416-429 (1970).
R. LANDESMAN
213
Landesman, R. and P.R. Gross: Develop. Biol. 18,571-589 (1968). Landesman, R. and P.R. Gross: Develop. Biol. 19, 244-260 (1969). Loening, U.E., K.W. Jones and M~L.Birnstiel: J. Mol. Biol. 45,353-366 (1969). Manchester, K.L.: Biochem. Actions Hormones 1,267-281 (1970). Muramatsu, M. and T. Fujisawa: Biochem. Biophys. Acta 157,476-492 (1968). Shiokawa, K. and K. Yamana: Develop. Biol. 16, 389-406 (1967). Stearns R.N. and A.B. KosteUov: in: The Chemical Basis of Development, eds. W.D. McElroy and B. Glass (Johns Hopkins Press) (1958). Sueoka, N. and T.Y. Cheng: J. Mol. Biol. 4,161-172 (1962). Woodland, H.R. and J.B. Gurdon: J. Embryol. Exptl. Morphol. 19, 363-385 (1968). Zimmerman, E.F. and B.W. Holler: J. Mol. Biol. 23, 149-161 (1967).