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Deoxyadenylate-rich sequences in sea urchin DNA during early development
Cell Differentiation 3, 117-126 ( 1974). © North-Holland Publishing Comp any
DEOXYADENYLATE-RICH SEQUENCES IN SEA URCHIN DNA DURING EARLY DEVELOPMENT Leona C. FITZMAURICE* and Robert F. BAKER** Department of Biological Sciences, University of Southern California, Los Angeles, California 90007, U.S.A. Accepted 20 March 1974
Sea urchin (Strongylocentrotus purpuratus) DNAs from morula and blastula stage embryos and from sperm were examined, by hybridization to [3H]polyuridylic acid, for the proportion of DNA sequences rich in deoxyadenylate. By this criterion, it was found that, relative to sperm DNA, morula DNA has 65% less deoxyadenylate-rich DNA. Blastula DNA has 1.7 times as much deoxyadenylaterich sequence as does morula DNA. Fractionation of these DNAs on neutral and alkaline cesium chloride density gradients indicates that the differences in the deoxyadenylate-rich content of the DNAs pertains predominantly to main density band DNA. Fractionation on cesium salt gradients also obviates the possibility that the measured differences in the DNAs result from preferential loss of satellite DNA during isolation of bulk DNAs.
The aim of this study was to examine the dA enriched regions in DNAs from morula and blastula stage embryos and from sperm of the sea urchin, Strongylocentrotus purpuratus. Regions enriched for dA in these DNAs were examined for several reasons, a) The proportion of the DNA enriched for one nucleotide-pair is amenable to study by hybridization with labeled polyribonucleotides (Kubinski et al., 1966; Hradecna et al., 1967; Shenkin et al., 1972). b) Regulatory regions in DNA have been suggested to be enriched for dA,T (lac operon (Lin et al., 1972)) or dG,dC (histone binding sites in calf thymus DNA (Clark et al., 1972)). c) Uridylaterich sequences have been observed within heterogeneous nuclear RNA molecules (Burdon et al., 1972; Dina et al., 1973). d) Electron microscopic studies of partially denatured Chinese hamster cell and chick fibroblast DNA have revealed that small, * Present address: Biochemistry Division, National Institute for Medical Research, Mill Hill, London NW7 1AA, England. ** To whom reprint requests should be addressed. Abbreviations: SSC = 0.15 M NaC1, 15 mM sodium citrate; dA = deoxyadenylate; dC = deoxycytidylate; dG = deoxyguanylate; T = thymidylate; poly (C) = polycytidylic acid; poly (U) = polyuridylic acid.
118
L.C. FITZMAURICEand R.F. BAKER
easily denatured regions (probably enriched for dA, T (Taylor et al., 1970)) are distributed throughout chromosomal DNA (Evenson et al., 1972). e) Earlier studies (Rosenkranz et al., 1967; Baker, 1972; Fitzmaurice et al., 1972)had indicated that there may be changes in the nuclear DNA of the sea urchin during development.
METHODS AND MATERIALS
Preparation of sperm and whole-cell DNA s S. purpurates sperm DNA was prepared and embryos were cultured as described earlier (Fitzmaurice et al., 1972). At a chosen stage of development, cultures were chilled, pelleted, and washed three times with 0.55 M KC1. The embryos were resuspended in two volumes of 0.15 M NaC1, 15 mM sodium citrate, 50 mM EDTA, 50 mM Tris, pH 8.4 measured at 5°C. An equal volume of water-saturated phenol was added along with sodium lauryl sulfate to 0.5%. The mixture was swirled at room temperature for 15-20 min, then centrifuged at 8000g for 10 rain. The aqueous phase and interface were removed and mixed with two volumes of ethanol (-20°C). The resulting precipitate was spooled and centrifuged out of solution. After three ethanol precipitations, the resulting precipitate was resuspended in the above buffer, and pronase (Calbiochem, preincubated for 2 hr at 37°C in the same buffer) was added to 1 mg/ml final concentration. This mixture was incubated overnight at 37°C; pancreatic ribonuclease (Worthington, in 0.15 M NaC1 and previously heated to 85°C for 20 rain) was then added to a final concentration of 20/ag/ml, and the mixture was incubated for 1 hr at 37°C. The DNA preparation was then phenol-extracted twice, ethanol precipitated three times, and dissolved and stored in 1 × SSC. A26o/A28o for all preparations was between 1.80 and 1.98. CsCl density gradient fractionation of DNA s Neutral CsC1 gradients were prepared following the general technique of Birnstiel et al. (1968). The preparation of alkaline CsC1 density gradients was essentially that of Flamm et al. (1967). Both neutral and alkaline gradients were centrifuged at 33,000 rev/min for 70 hr in a Spinco 50 ti rotor, and fractions of approximately 0.1 ml (6-drop) were collected. The pH of the alkaline fractions was adjusted to pH 7.0 with 1 N HC1; all fractions were brought to a final volume of 1 ml with the addition of 0.1 X SSC prior to reading the absorbance of 260 nm. Hybridization of DNA with [3H] polyribonucleotides The assay tubes (as described in the figure legends) were sealed and, in all cases, except as noted in fig. 1, heated in a boiling water bath for 15 min. The tubes were then placed in a 20°C water bath for 4 hr. At the end of this time, pancreatic ribonuclease (Worthington, in 0.15 M NaC1 and previously heated to 85°C for 20 min) was added to 5/ag/ml to each tube, and the mixture was held at room temperature for 30 min to 1 hr. These conditions used for digestion of the [3HI_
Deoxyadenylate-rich sequences in DNA
119
poly (U) : DNA hybrids have been shown to completely hydrolyze non-hybridized [3 H] poly (U). Hybrids were collected on Schleicher and Schuell B6 24 mm nitrocellulose filters (previously soaked in boiling 2 X SSC for 30 min, rinsed, and washed with 20 ml 2 × SSC). The filters were then washed with 100 ml of 2 X SSC, dried, and counted. Parallel sets of assay tubes lacking only DNA were used to produce blank values which were subtracted from experimental values. [ 3H] polyribonucleo tides [3H]poly (U) (82.2 mCi/mM phosphorous, 4.2/lg//aCi) a n d [3H] poly (C) (44.8 mCi/mM phosphorous, 8.95/ag//aCi) were purchased from Miles Laboratories, Inc., Kankakee, Illinois.
RESULTS AND DISCUSSION S. purpurates whole-cell DNAs from morula and blastula stage embryos and sperm DNA were examined for their ability to hybridize [3H] poly (U) in satura-
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0.1 0"2 03 0.4 0"5 3H-POLYURIDYLIC ACID (ug) Fig. 1. Hybridization of sperm, whole-cell morula, and whole-cell blastula DNAs from Strongylocentrotus purpuratus with increasing amounts of [3H]polyuridylic acid: comparison of methods. For each point on a saturation curve, a hybridization tube was prepared containing 1 ug of DNA plus a quantity of [3H] polyuridylic acid as indicated on the abscissa of the graph. The hybridization reaction was in a total volume of 1 ml 2 × SSC, and the procedure followed was as described in Methods and Materials. For one set of curves, the DNA and the [3H] polyuridylic acid were combined in hybridization tubes prior to denaturation of the DNA by boiling. For the other set of curves, the DNA was first denatured by boiling and then combined with the [3H] polyuridylic acid in the hybridization tubes. A,L whole-cell morula DNA; e,o whole-cell blastula DNA; v,v sperm DNA. Closed symbols indicate [3HI polyuridylic acid present during denaturation; open symbols indicate [3 H] polyuridylic acid combined after denaturation.
315 110 185
Cpm per #g DNA
0.26 0.09 0.15
(%)
3149/.209 893/.101 1265/.104
Amount Cpm/A260 hybridizationa
300 179 243
Cpm per ~ DNA U
0.25 0.15 0.20
(%) 2450/.163 1211/.160 2814/.243
Amount Cpm/A260 hybridization a
378 189 290
Cpm per/~ DNA u
0.32 0.16 0.24
(%) 1574/.209 935/.101 907/.104
Amount Cpm/A260 hybridization a
150 187 181
Cpm per ~ DNA u
0.25 0.31 0.30
(~)
Amount hybridization a
Neutral CsC1 gradients (figs, 2, 3 and 4)
Alkaline CsCI gradients (figs, 5, 6, and 7)
Saturation hybridization (fig. 1)
Neutral CsC1 gradients (figs. 2, 3, and 4)
Cpm [3HI poly (C) hybridized
Cpm [3H] poly (U) hybridized
a The amount hybridization was calculated on the basis that [3H] polyuridylic acid used had a specific activity of 120,000 cpm/~g and [3 HI polycytidylic acid used had a specific activity of 60,000 cpm/tzg. The labeled polyribonucleotides were assumed to hybridize to the single-stranded DNA molecules in a 1 : 1 ratio. b The DNA concentration in the peak fraction from the absorbance profile was calculated on the basis that 1.0 absorbance unit at 260 nm is equal to 50 #g double-stranded DNA per ml or 40 ug denatured (single-stranded) DNA per ml.
Sperm Morula Blastula
DNA
Table 1 Hybridization of [3H] polyribonucleotides to absorbance peak fraction (main band DNAs) from neutral and alkaline cesium chloride gradients; comparison with saturation hybridization plateau values derived from unfractionated DNAs.
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Fig. 2. Hybridization of [3H]polyuridylic acid and [3H]polycytidylic acid to neutral CsC1 gradient fractions of Strongylocentrotus purpuratus sperm DNA. For hybridization, 0.3 ml was removed from each diluted fraction and combined with 1 pg [3H]polyuridylic acid or [3H]polycytidylic acid in 0.7 ml 3 X SSC. Hybridization reactions were as described in Methods and Materials. ~, absorbance at 260 nm; o, [3H] polyuridylic acid hybridized; o, [3H] polycytidylic acid hybridized.
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Fig. 3. Hybridization ot [3H]polyuridylic acid and [3H]polycytidylic acid to neutral CsCI gradient fractions of Strongylocentrotus purpuratus morula DNA. The procedure was as described in fig. 2. a, absorbance at 260 nm; e, [3H] polyuridylic acid hybridized; o, [3H] polycytidylic acid hybridized.
122
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Fig. 4. Hybridization of [3H] polyuridylic and [3H] polycytidylic acid to neutral CsC1 gradient fractions of Strongylocentrotus purpuratus blastula DNA. The procedure was as described in fig. 2. n, absorbance at 260 nm; e, [3H] polyuridylic acid hybridized; o, [3H] polycytidylic acid hybridized.
tion hybridization experiments. The hybridization technique of Shenkin et al. (1972) was slightly modified for these experiments: DNA was denatured by boiling in the presence of the labeled polyribonucleotide. The hybridization reaction was then allowed to proceed at the optimum temperature of 20°C, in solution, with maximum hybridization being obtained in the 4 hr reaction time used here (Shenkin et al., 1972). Lower saturation plateaus were obtained when labelled polyribonucleotides were added after denaturation of the DNA (fig. 1). As can be seen in fig. 1, sperm DNA saturates at a much higher level (315 counts/min//ag DNA) than does whole-cell morula DNA (110 counts/min//ag DNA) with whole-cell blastula DNA falling in between (185 counts/min/lag DNA). The saturation plateau levels are summarized in table 1. Assuming that poly (U) hybridized to poly d(A) regions in the DNA in a 1:1 ratio, the percentage of the denatured DNA hybridizing was also calculated (table I). Since poly (U) has been observed to hybridize to poly d(A) in a ratio of 2 : 1 under some conditions (Riley et al., 1966), it is possible that these estimates are high, perhaps by as much as a factor of two. Also, there may be as many as 10% mismatched base-pairs in the [aH]poly (U) : eukaryotic DNA hybrids (McCarthy et al., 1970; Shenkin et al., 1972); thus, the [a H] poly (U) hybridization is measuring poly d(A) regions which may contain a low percentage of other nucleotides. This saturation hybridization data suggests several things. First, there is a decrease in the proportion of poly d(A)-rich DNA during the first few cleavages following fertilization. Even if egg DNA contained no poly d(A)-rich regions, upon fertilization the proportion of zygote nuclear DNA homologous to poly (U) should
Deoxyadenylate-rich sequences in DNA
123
be reduced only to 50% of the amount measured in sperm DNA. The relative proportion of poly d(A)-rich material present in morula DNA as measured by this hybridization method is, however, 65% less than the amount of material present in sperm DNA. Second, there is an increase between morula and blastula stages in the proportion of DNA hybridizing to poly(U) (an increase of approx. 1.7 times). Both of these conclusions could be unwarrented for genomic DNA, however, since whole-cell DNAs were used to produce two of the DNA saturation curves. One possibility is that the observed changes might result from variations in satellite DNA content in vivo (Travaglini et al., 1972), or as a result of selective loss of satellite DNAs during the DNA isolation procedure. It is also possible that cytoplasmic (mitochondrial) DNA is diluting the hybridizable nuclear DNA sequences. However, at the morula stage, only about 0.8% (0.2% at the blastula stage) of the total embryo DNA is represented by mitochondrial DNA (Piko et al., 1967; Piko, 1969). To examine these possibilities, the three DNA preparations were fractionated on neutral CsC1 density gradients. This technique has been shown to separate sea urchin mitochondrial DNA from nuclear (Piko et al., 1967, 1968) and to separate density satellite DNAs from main band DNAs (Birnstiel et al., 1968). Figs. 2 - 4 show results of hybridization of [3H]poly (U) to gradient fractions of sperm, morula, and blastula DNAs. It is apparent that most of the hybridization of the homopolymer is to main density band DNA. Mitochondrial DNA (which, for S. purpuratus, has a buoyant density of 1.701 g/cc in neutral CsC1 (Piko et al., 1968) would appear to the left of the main band DNA (i.e., towards the bottom of the gradients). Neither an absorbance peak nor a hybridization peak are apparent in the sperm and blastula DNAs. A dispersed region of absorbance in regions of higher density is seen in morula DNA, but no significant hybridization is observed. There is also the suggestion of a small quantity of lighter density satellite in the sperm DNA and morula DNA. The percentage of hybridization of [3 H] poly (U) to the absorbance peak fraction of the main density band of a particular stage DNA is in relatively close agreement with the saturation plateau value of the unfractionated whole-cell DNA (table 1). Thus, it appears that any selective loss of satellite DNAs in isolating bulk DNAs has essentially not altered the quantitation of relative amounts of polydeoxyadenylate-rich sequence in stage-specific bulk DNAs. [3H]poly (C) was also hybridized to fractions of the neutral CsC1 gradients (figs. 2-4). It can be seen that the absorbance peak hybridization values (table 1) for [3H]poly (C) are not proportional to those produced by [3H]poly (U) in comparing sperm, morula, and blastula DNAs. This suggests that [3H]poly (U) hybridizes to the DNA, as fractionated on neutral CsC1 gradients, in a manner specific for this particular polyribonucleotide. DNAs were further examined for satellite content on alkaline CsC1 gradients (figs. 5-7). The alkaline condition used here should destroy any polyadenylic acid contamination which would be resistant (Darnell et al., 1971; Lee et al., 1971)to
124
L.C. F I T Z M A U R I C E and R.F. BAKER I
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Fig. 5. Hybridization o f [3 H] polyuridylic acid to alkaline CsC1 gradient fractions of Strongylocentrotus purpuratus sperm DNA. For hybridization, 0.3 ml was removed from each diluted fraction (pH 7.0) and combined with 1 gg [3HI polyuridylic acid in 0.7 ml 3 × SSC. Hybridization reactions were as described in Methods and Materials. ~, absorbance at 260 n m ; e, [3H]polyuridylic acid hybridized.
the ribonuclease used in preparing the DNAs. Most of the polyribonucleotide hybridization is to main band DNAs. As shown in table I, the hybridization percentages of the peak fractions of main band DNAs from the alkaline CsC1 gradients are also in relatively good agreement with the saturation hybridization values obtained in fig. 1. In sum, neutral and alkaline CsC1 density gradient data supports the contention that the dA-rich content of the three sea urchin DNAs examined is different and that the differences pertain predominantly to the main density band DNA. f
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Fig. 6. Hybridization of [ 3 H ] polyuridylic acid to alkaline CsCI gradient fractions o f Strongylocentrotus purpuratus morula DNA. The procedure was as described in fig. 5. ~ absorbance at 260 n m ; e, [3H] polyuridylic acid hybridized.
Deoxyadenylate-rieh sequences in DNA i
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Fig. 7. Hybridization of [3 H] polyuridylic acid to alkaline CsC1 gradient fractions of Strongylocentrotus purpurates blastula DNA. The procedure was as described in fig. 5. a, absorbance at 260 nm; o, [3H] polyuridylic acid hybridized.
ACKNOWLEDGMENTS This work was supported by a grant (HDO4015) from the National Institutes of Health and an institutional Biomedical Sciences Support grant from the same agency. L.C.F. was a National Science Foundation predoctoral trainee.
REFERENCES Baker, R.F.: J. Cell Sci. 1 1 , 1 5 3 - 1 7 1 (1972). Birnstiel, M., J. Speirs, I. Purdom, K. Jones and U.E. Loening: Nature, Lond. 219, 4 5 4 - 4 6 3 (1968). Burdon, R.H. and A. Shenkin: Fed. Eur. Biochem. Soc. Letters 24, 1 1 - 1 4 (1972). Clark, R.J. and G. Felsenfeld: Nature New Biol. 2 4 0 , 2 2 6 - 2 2 9 (1972). Darnell, J.E., R. Wall and R.J. Tushinski: Proc. Natl. Acad. Sci. U.S. 68, 1 3 2 1 - 1 3 2 5 (1971). Dina, D., M. Crippa and E. Beccari: Nature New Biol. 2 4 2 , 1 0 1 - 1 0 5 (1973). Evenson, D.P., W.A. Mego and J.H. Taylor: Chromosoma 3 9 , 2 2 5 - 2 3 5 (1972). Fitzmaurice, L.C. and R.F. Baker: Biochim. Biophys. Acta 2 7 2 , 5 1 0 - 5 1 7 (1972). Flamm, W.G., M. McCallum and P.M.B. Walker: Proc. Natl. Acad. Sci. U.S. 57, 1729-1734 (1967). Hradecna, Z. and W. Szybalski: Virology 3 2 , 6 3 3 - 6 4 3 (1967). Kubinski, H., Z. Opara-Kubinska and W. Szybalski: J. Mol. Biol. 20, 3 1 3 - 3 2 9 (1966). Lee, S.Y., J. Mendecki and G. Brawerman: Proc. Natl. Acad. Sci. U.S. 68, 1331-1335 (1971). Lin, S.-Y. and A.D. Riggs: Proc. Natl. Acad. Sci. U.S. 69, 2 5 7 4 - 2 5 7 6 (1972). McCarthy, B.J. and R.B. Church: Ann. Rev. Bioehem. 3 9 , 1 3 1 - 1 5 0 (1970). Piko, L.: Zool. 9, 1118 (1969). Piko, L., D.G. Blair, A. Tyler and J. Vinograd: Proc. Natl. Acad. Sci. U.S. 5 9 , 8 3 8 - 8 4 5 (1968). Piko, L., A. Tyler and J. Vinograd: Biol. Bull. 132, 6 8 - 9 0 (1967). Riley, M., B. Maling and M.J. Chamberlin: J. Mol. Biol. 20, 3 5 9 - 3 8 9 (1966).
126
L.C. FITZMAURICE and R.t:. BAKI.,.R
Rosenkranz, H.S. and G.A. Carden Iil: Nature, Lond. 213, 1024 1025 (1967). Shenkin, A. and R.H. Burdon: Fed. Eur. Biochem. Soc. Letters 2 2 , 1 5 7 - 1 6 0 (1972). Taylor, J.H., W.A. Mego and D.P. Evenson: In: The Neurosciences: Second Study Program, cd. F.O. Schmitt (The Rockefeller University Press, New York) pp. 9 9 8 - 1 0 1 3 (1970). Travaglini, E.C., J. Petrovic and J. Schultz: Genetics 7 2 , 4 1 9 - 4 3 0 (1972).
DEOXYADENYLATE-RICH SEQUENCES IN SEA URCHIN DNA DURING EARLY DEVELOPMENT Leona C. FITZMAURICE* and Robert F. BAKER** Department of Biological Sciences, University of Southern California, Los Angeles, California 90007, U.S.A. Accepted 20 March 1974
Sea urchin (Strongylocentrotus purpuratus) DNAs from morula and blastula stage embryos and from sperm were examined, by hybridization to [3H]polyuridylic acid, for the proportion of DNA sequences rich in deoxyadenylate. By this criterion, it was found that, relative to sperm DNA, morula DNA has 65% less deoxyadenylate-rich DNA. Blastula DNA has 1.7 times as much deoxyadenylaterich sequence as does morula DNA. Fractionation of these DNAs on neutral and alkaline cesium chloride density gradients indicates that the differences in the deoxyadenylate-rich content of the DNAs pertains predominantly to main density band DNA. Fractionation on cesium salt gradients also obviates the possibility that the measured differences in the DNAs result from preferential loss of satellite DNA during isolation of bulk DNAs.
The aim of this study was to examine the dA enriched regions in DNAs from morula and blastula stage embryos and from sperm of the sea urchin, Strongylocentrotus purpuratus. Regions enriched for dA in these DNAs were examined for several reasons, a) The proportion of the DNA enriched for one nucleotide-pair is amenable to study by hybridization with labeled polyribonucleotides (Kubinski et al., 1966; Hradecna et al., 1967; Shenkin et al., 1972). b) Regulatory regions in DNA have been suggested to be enriched for dA,T (lac operon (Lin et al., 1972)) or dG,dC (histone binding sites in calf thymus DNA (Clark et al., 1972)). c) Uridylaterich sequences have been observed within heterogeneous nuclear RNA molecules (Burdon et al., 1972; Dina et al., 1973). d) Electron microscopic studies of partially denatured Chinese hamster cell and chick fibroblast DNA have revealed that small, * Present address: Biochemistry Division, National Institute for Medical Research, Mill Hill, London NW7 1AA, England. ** To whom reprint requests should be addressed. Abbreviations: SSC = 0.15 M NaC1, 15 mM sodium citrate; dA = deoxyadenylate; dC = deoxycytidylate; dG = deoxyguanylate; T = thymidylate; poly (C) = polycytidylic acid; poly (U) = polyuridylic acid.
118
L.C. FITZMAURICEand R.F. BAKER
easily denatured regions (probably enriched for dA, T (Taylor et al., 1970)) are distributed throughout chromosomal DNA (Evenson et al., 1972). e) Earlier studies (Rosenkranz et al., 1967; Baker, 1972; Fitzmaurice et al., 1972)had indicated that there may be changes in the nuclear DNA of the sea urchin during development.
METHODS AND MATERIALS
Preparation of sperm and whole-cell DNA s S. purpurates sperm DNA was prepared and embryos were cultured as described earlier (Fitzmaurice et al., 1972). At a chosen stage of development, cultures were chilled, pelleted, and washed three times with 0.55 M KC1. The embryos were resuspended in two volumes of 0.15 M NaC1, 15 mM sodium citrate, 50 mM EDTA, 50 mM Tris, pH 8.4 measured at 5°C. An equal volume of water-saturated phenol was added along with sodium lauryl sulfate to 0.5%. The mixture was swirled at room temperature for 15-20 min, then centrifuged at 8000g for 10 rain. The aqueous phase and interface were removed and mixed with two volumes of ethanol (-20°C). The resulting precipitate was spooled and centrifuged out of solution. After three ethanol precipitations, the resulting precipitate was resuspended in the above buffer, and pronase (Calbiochem, preincubated for 2 hr at 37°C in the same buffer) was added to 1 mg/ml final concentration. This mixture was incubated overnight at 37°C; pancreatic ribonuclease (Worthington, in 0.15 M NaC1 and previously heated to 85°C for 20 rain) was then added to a final concentration of 20/ag/ml, and the mixture was incubated for 1 hr at 37°C. The DNA preparation was then phenol-extracted twice, ethanol precipitated three times, and dissolved and stored in 1 × SSC. A26o/A28o for all preparations was between 1.80 and 1.98. CsCl density gradient fractionation of DNA s Neutral CsC1 gradients were prepared following the general technique of Birnstiel et al. (1968). The preparation of alkaline CsC1 density gradients was essentially that of Flamm et al. (1967). Both neutral and alkaline gradients were centrifuged at 33,000 rev/min for 70 hr in a Spinco 50 ti rotor, and fractions of approximately 0.1 ml (6-drop) were collected. The pH of the alkaline fractions was adjusted to pH 7.0 with 1 N HC1; all fractions were brought to a final volume of 1 ml with the addition of 0.1 X SSC prior to reading the absorbance of 260 nm. Hybridization of DNA with [3H] polyribonucleotides The assay tubes (as described in the figure legends) were sealed and, in all cases, except as noted in fig. 1, heated in a boiling water bath for 15 min. The tubes were then placed in a 20°C water bath for 4 hr. At the end of this time, pancreatic ribonuclease (Worthington, in 0.15 M NaC1 and previously heated to 85°C for 20 min) was added to 5/ag/ml to each tube, and the mixture was held at room temperature for 30 min to 1 hr. These conditions used for digestion of the [3HI_
Deoxyadenylate-rich sequences in DNA
119
poly (U) : DNA hybrids have been shown to completely hydrolyze non-hybridized [3 H] poly (U). Hybrids were collected on Schleicher and Schuell B6 24 mm nitrocellulose filters (previously soaked in boiling 2 X SSC for 30 min, rinsed, and washed with 20 ml 2 × SSC). The filters were then washed with 100 ml of 2 X SSC, dried, and counted. Parallel sets of assay tubes lacking only DNA were used to produce blank values which were subtracted from experimental values. [ 3H] polyribonucleo tides [3H]poly (U) (82.2 mCi/mM phosphorous, 4.2/lg//aCi) a n d [3H] poly (C) (44.8 mCi/mM phosphorous, 8.95/ag//aCi) were purchased from Miles Laboratories, Inc., Kankakee, Illinois.
RESULTS AND DISCUSSION S. purpurates whole-cell DNAs from morula and blastula stage embryos and sperm DNA were examined for their ability to hybridize [3H] poly (U) in satura-
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0.1 0"2 03 0.4 0"5 3H-POLYURIDYLIC ACID (ug) Fig. 1. Hybridization of sperm, whole-cell morula, and whole-cell blastula DNAs from Strongylocentrotus purpuratus with increasing amounts of [3H]polyuridylic acid: comparison of methods. For each point on a saturation curve, a hybridization tube was prepared containing 1 ug of DNA plus a quantity of [3H] polyuridylic acid as indicated on the abscissa of the graph. The hybridization reaction was in a total volume of 1 ml 2 × SSC, and the procedure followed was as described in Methods and Materials. For one set of curves, the DNA and the [3H] polyuridylic acid were combined in hybridization tubes prior to denaturation of the DNA by boiling. For the other set of curves, the DNA was first denatured by boiling and then combined with the [3H] polyuridylic acid in the hybridization tubes. A,L whole-cell morula DNA; e,o whole-cell blastula DNA; v,v sperm DNA. Closed symbols indicate [3HI polyuridylic acid present during denaturation; open symbols indicate [3 H] polyuridylic acid combined after denaturation.
315 110 185
Cpm per #g DNA
0.26 0.09 0.15
(%)
3149/.209 893/.101 1265/.104
Amount Cpm/A260 hybridizationa
300 179 243
Cpm per ~ DNA U
0.25 0.15 0.20
(%) 2450/.163 1211/.160 2814/.243
Amount Cpm/A260 hybridization a
378 189 290
Cpm per/~ DNA u
0.32 0.16 0.24
(%) 1574/.209 935/.101 907/.104
Amount Cpm/A260 hybridization a
150 187 181
Cpm per ~ DNA u
0.25 0.31 0.30
(~)
Amount hybridization a
Neutral CsC1 gradients (figs, 2, 3 and 4)
Alkaline CsCI gradients (figs, 5, 6, and 7)
Saturation hybridization (fig. 1)
Neutral CsC1 gradients (figs. 2, 3, and 4)
Cpm [3HI poly (C) hybridized
Cpm [3H] poly (U) hybridized
a The amount hybridization was calculated on the basis that [3H] polyuridylic acid used had a specific activity of 120,000 cpm/~g and [3 HI polycytidylic acid used had a specific activity of 60,000 cpm/tzg. The labeled polyribonucleotides were assumed to hybridize to the single-stranded DNA molecules in a 1 : 1 ratio. b The DNA concentration in the peak fraction from the absorbance profile was calculated on the basis that 1.0 absorbance unit at 260 nm is equal to 50 #g double-stranded DNA per ml or 40 ug denatured (single-stranded) DNA per ml.
Sperm Morula Blastula
DNA
Table 1 Hybridization of [3H] polyribonucleotides to absorbance peak fraction (main band DNAs) from neutral and alkaline cesium chloride gradients; comparison with saturation hybridization plateau values derived from unfractionated DNAs.
N
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Fig. 2. Hybridization of [3H]polyuridylic acid and [3H]polycytidylic acid to neutral CsC1 gradient fractions of Strongylocentrotus purpuratus sperm DNA. For hybridization, 0.3 ml was removed from each diluted fraction and combined with 1 pg [3H]polyuridylic acid or [3H]polycytidylic acid in 0.7 ml 3 X SSC. Hybridization reactions were as described in Methods and Materials. ~, absorbance at 260 nm; o, [3H] polyuridylic acid hybridized; o, [3H] polycytidylic acid hybridized.
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Fig. 3. Hybridization ot [3H]polyuridylic acid and [3H]polycytidylic acid to neutral CsCI gradient fractions of Strongylocentrotus purpuratus morula DNA. The procedure was as described in fig. 2. a, absorbance at 260 nm; e, [3H] polyuridylic acid hybridized; o, [3H] polycytidylic acid hybridized.
122
L.C. FITZMAURICE and R.F. BAKER i
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Fig. 4. Hybridization of [3H] polyuridylic and [3H] polycytidylic acid to neutral CsC1 gradient fractions of Strongylocentrotus purpuratus blastula DNA. The procedure was as described in fig. 2. n, absorbance at 260 nm; e, [3H] polyuridylic acid hybridized; o, [3H] polycytidylic acid hybridized.
tion hybridization experiments. The hybridization technique of Shenkin et al. (1972) was slightly modified for these experiments: DNA was denatured by boiling in the presence of the labeled polyribonucleotide. The hybridization reaction was then allowed to proceed at the optimum temperature of 20°C, in solution, with maximum hybridization being obtained in the 4 hr reaction time used here (Shenkin et al., 1972). Lower saturation plateaus were obtained when labelled polyribonucleotides were added after denaturation of the DNA (fig. 1). As can be seen in fig. 1, sperm DNA saturates at a much higher level (315 counts/min//ag DNA) than does whole-cell morula DNA (110 counts/min//ag DNA) with whole-cell blastula DNA falling in between (185 counts/min/lag DNA). The saturation plateau levels are summarized in table 1. Assuming that poly (U) hybridized to poly d(A) regions in the DNA in a 1:1 ratio, the percentage of the denatured DNA hybridizing was also calculated (table I). Since poly (U) has been observed to hybridize to poly d(A) in a ratio of 2 : 1 under some conditions (Riley et al., 1966), it is possible that these estimates are high, perhaps by as much as a factor of two. Also, there may be as many as 10% mismatched base-pairs in the [aH]poly (U) : eukaryotic DNA hybrids (McCarthy et al., 1970; Shenkin et al., 1972); thus, the [a H] poly (U) hybridization is measuring poly d(A) regions which may contain a low percentage of other nucleotides. This saturation hybridization data suggests several things. First, there is a decrease in the proportion of poly d(A)-rich DNA during the first few cleavages following fertilization. Even if egg DNA contained no poly d(A)-rich regions, upon fertilization the proportion of zygote nuclear DNA homologous to poly (U) should
Deoxyadenylate-rich sequences in DNA
123
be reduced only to 50% of the amount measured in sperm DNA. The relative proportion of poly d(A)-rich material present in morula DNA as measured by this hybridization method is, however, 65% less than the amount of material present in sperm DNA. Second, there is an increase between morula and blastula stages in the proportion of DNA hybridizing to poly(U) (an increase of approx. 1.7 times). Both of these conclusions could be unwarrented for genomic DNA, however, since whole-cell DNAs were used to produce two of the DNA saturation curves. One possibility is that the observed changes might result from variations in satellite DNA content in vivo (Travaglini et al., 1972), or as a result of selective loss of satellite DNAs during the DNA isolation procedure. It is also possible that cytoplasmic (mitochondrial) DNA is diluting the hybridizable nuclear DNA sequences. However, at the morula stage, only about 0.8% (0.2% at the blastula stage) of the total embryo DNA is represented by mitochondrial DNA (Piko et al., 1967; Piko, 1969). To examine these possibilities, the three DNA preparations were fractionated on neutral CsC1 density gradients. This technique has been shown to separate sea urchin mitochondrial DNA from nuclear (Piko et al., 1967, 1968) and to separate density satellite DNAs from main band DNAs (Birnstiel et al., 1968). Figs. 2 - 4 show results of hybridization of [3H]poly (U) to gradient fractions of sperm, morula, and blastula DNAs. It is apparent that most of the hybridization of the homopolymer is to main density band DNA. Mitochondrial DNA (which, for S. purpuratus, has a buoyant density of 1.701 g/cc in neutral CsC1 (Piko et al., 1968) would appear to the left of the main band DNA (i.e., towards the bottom of the gradients). Neither an absorbance peak nor a hybridization peak are apparent in the sperm and blastula DNAs. A dispersed region of absorbance in regions of higher density is seen in morula DNA, but no significant hybridization is observed. There is also the suggestion of a small quantity of lighter density satellite in the sperm DNA and morula DNA. The percentage of hybridization of [3 H] poly (U) to the absorbance peak fraction of the main density band of a particular stage DNA is in relatively close agreement with the saturation plateau value of the unfractionated whole-cell DNA (table 1). Thus, it appears that any selective loss of satellite DNAs in isolating bulk DNAs has essentially not altered the quantitation of relative amounts of polydeoxyadenylate-rich sequence in stage-specific bulk DNAs. [3H]poly (C) was also hybridized to fractions of the neutral CsC1 gradients (figs. 2-4). It can be seen that the absorbance peak hybridization values (table 1) for [3H]poly (C) are not proportional to those produced by [3H]poly (U) in comparing sperm, morula, and blastula DNAs. This suggests that [3H]poly (U) hybridizes to the DNA, as fractionated on neutral CsC1 gradients, in a manner specific for this particular polyribonucleotide. DNAs were further examined for satellite content on alkaline CsC1 gradients (figs. 5-7). The alkaline condition used here should destroy any polyadenylic acid contamination which would be resistant (Darnell et al., 1971; Lee et al., 1971)to
124
L.C. F I T Z M A U R I C E and R.F. BAKER I
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Fig. 5. Hybridization o f [3 H] polyuridylic acid to alkaline CsC1 gradient fractions of Strongylocentrotus purpuratus sperm DNA. For hybridization, 0.3 ml was removed from each diluted fraction (pH 7.0) and combined with 1 gg [3HI polyuridylic acid in 0.7 ml 3 × SSC. Hybridization reactions were as described in Methods and Materials. ~, absorbance at 260 n m ; e, [3H]polyuridylic acid hybridized.
the ribonuclease used in preparing the DNAs. Most of the polyribonucleotide hybridization is to main band DNAs. As shown in table I, the hybridization percentages of the peak fractions of main band DNAs from the alkaline CsC1 gradients are also in relatively good agreement with the saturation hybridization values obtained in fig. 1. In sum, neutral and alkaline CsC1 density gradient data supports the contention that the dA-rich content of the three sea urchin DNAs examined is different and that the differences pertain predominantly to the main density band DNA. f
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Fig. 6. Hybridization of [ 3 H ] polyuridylic acid to alkaline CsCI gradient fractions o f Strongylocentrotus purpuratus morula DNA. The procedure was as described in fig. 5. ~ absorbance at 260 n m ; e, [3H] polyuridylic acid hybridized.
Deoxyadenylate-rieh sequences in DNA i
125 i
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Fig. 7. Hybridization of [3 H] polyuridylic acid to alkaline CsC1 gradient fractions of Strongylocentrotus purpurates blastula DNA. The procedure was as described in fig. 5. a, absorbance at 260 nm; o, [3H] polyuridylic acid hybridized.
ACKNOWLEDGMENTS This work was supported by a grant (HDO4015) from the National Institutes of Health and an institutional Biomedical Sciences Support grant from the same agency. L.C.F. was a National Science Foundation predoctoral trainee.
REFERENCES Baker, R.F.: J. Cell Sci. 1 1 , 1 5 3 - 1 7 1 (1972). Birnstiel, M., J. Speirs, I. Purdom, K. Jones and U.E. Loening: Nature, Lond. 219, 4 5 4 - 4 6 3 (1968). Burdon, R.H. and A. Shenkin: Fed. Eur. Biochem. Soc. Letters 24, 1 1 - 1 4 (1972). Clark, R.J. and G. Felsenfeld: Nature New Biol. 2 4 0 , 2 2 6 - 2 2 9 (1972). Darnell, J.E., R. Wall and R.J. Tushinski: Proc. Natl. Acad. Sci. U.S. 68, 1 3 2 1 - 1 3 2 5 (1971). Dina, D., M. Crippa and E. Beccari: Nature New Biol. 2 4 2 , 1 0 1 - 1 0 5 (1973). Evenson, D.P., W.A. Mego and J.H. Taylor: Chromosoma 3 9 , 2 2 5 - 2 3 5 (1972). Fitzmaurice, L.C. and R.F. Baker: Biochim. Biophys. Acta 2 7 2 , 5 1 0 - 5 1 7 (1972). Flamm, W.G., M. McCallum and P.M.B. Walker: Proc. Natl. Acad. Sci. U.S. 57, 1729-1734 (1967). Hradecna, Z. and W. Szybalski: Virology 3 2 , 6 3 3 - 6 4 3 (1967). Kubinski, H., Z. Opara-Kubinska and W. Szybalski: J. Mol. Biol. 20, 3 1 3 - 3 2 9 (1966). Lee, S.Y., J. Mendecki and G. Brawerman: Proc. Natl. Acad. Sci. U.S. 68, 1331-1335 (1971). Lin, S.-Y. and A.D. Riggs: Proc. Natl. Acad. Sci. U.S. 69, 2 5 7 4 - 2 5 7 6 (1972). McCarthy, B.J. and R.B. Church: Ann. Rev. Bioehem. 3 9 , 1 3 1 - 1 5 0 (1970). Piko, L.: Zool. 9, 1118 (1969). Piko, L., D.G. Blair, A. Tyler and J. Vinograd: Proc. Natl. Acad. Sci. U.S. 5 9 , 8 3 8 - 8 4 5 (1968). Piko, L., A. Tyler and J. Vinograd: Biol. Bull. 132, 6 8 - 9 0 (1967). Riley, M., B. Maling and M.J. Chamberlin: J. Mol. Biol. 20, 3 5 9 - 3 8 9 (1966).
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Rosenkranz, H.S. and G.A. Carden Iil: Nature, Lond. 213, 1024 1025 (1967). Shenkin, A. and R.H. Burdon: Fed. Eur. Biochem. Soc. Letters 2 2 , 1 5 7 - 1 6 0 (1972). Taylor, J.H., W.A. Mego and D.P. Evenson: In: The Neurosciences: Second Study Program, cd. F.O. Schmitt (The Rockefeller University Press, New York) pp. 9 9 8 - 1 0 1 3 (1970). Travaglini, E.C., J. Petrovic and J. Schultz: Genetics 7 2 , 4 1 9 - 4 3 0 (1972).