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Localization of amplified ribosomal DNA in the oocyte of Xenopus laevis
274
PKELIMINARY NOTES
PRELIMINARY NOTES BBA 91224 Lo~liw~tion of arnplified ribosomol D N A in the oo~/te of Xenopus I ~ l s There are some z5en nucleoli I in the ooeyte nucleus of Xenopus laevis, the south african clawed toad, compared to one or two in somatic nuclei. The oocyte nuclsoli contain DNA which has arisen by gene amplification (of. ref. z). It hasbeen shown that in somatic coils the nuclcolus is formed largely through the activity of the ribosomal cistrons which are clustered together at the nucleolar organiser (c/. ref. 3). These ribosomal cistroas may be isolated from somatic tissues in a DN'A satellite (rDNA) of a buoyant density of L723 g ' c m -s (refs. 4. 5). In hybridization experiments with pure :DNA it may be demonstrated that 35 % of the satellite mass, and possible more, is made up from ~8-S and x8-S cistruns 0. We have now estimated the mass and buoyant density of the rDNA contained in nncleoli isolated from immature ocoytes of X. laevls. Ovarian tissue from 6-8 Xenopus females was disaggregated by treatment with pronase (aoopg/ml in 0,08 M KCI; 0,02 M ~aCI), The oocytes were fractionatod according to size by floatation ~ in o,o8 M KCI, o,o2 M NaCi and oocytes of overall diameter o,zu-o.45 ram, accompanied by some adhering follicle cells, were collected. This procedure yielded x.to4-2.xo 4 oucytes, The oocytes were suspended in 5 ml of o,4 M sucrose, containing 3 mM Ca¢I2, 0.o8 M KCI and o.oz M NaCI, and ruptured by rapid expulsion through a Pasteur pipette, The homogenate was filtered through three layers of cheese cloth and the nueleolipelleted through ~ M sucrose, 3 mM CaCI¢at zo ooo rev./min for 7 rain in a Spinco rotor SW 25.I at 4°° Light and electron microscopic examination of the preparation shewed the nudceli to be well preserved and free from yolk piatelets. They contained a small fraction of pigment granules and some follicle cell nuclei (<: z %), DNA was extracted according to ~t~ARMUR8, with the modification that after the initiat precipitation with ethar~ol the DIq'A was dissolved in o,25 b[ NaCI, o.oi5 M sodium citrate (pH 7.~), treated with ribonuelease (zoo pg/ml) and exhaustively diaiysed against the solvent at room temperature. The DNA was quantitatively pelleted by centrifugation for 2o h a~: 5o ooo rev./min (I5°) inaSpinco rotor No, 50, Equilibrium centrifugation in CsCl in the analytical centrifuge resolved the nucleolar DNA into two components banding in CsC1 at 1.7z9g'em -s and z.697 g'em -a (Fig, xe). (I,729) DNA was found to be hybridizable to rRNA, hence is identified as rDNA (Fig, zb), The (z.697) DNA which has the same density as somatic (total) nuclear DNA is thought to be derived from the contaminating follicle cell nuclei, each of which contributes some 6 pg DNA9, i,e, some 60o times more D1qA than does each oocyte nueleohis (see below). The DNA extracted from whole oocytes is resolved into three components banding at z,7~,9, z,723 and 1,7ox g,cm-s. The (z.7ox) componentis most likely a composite of the very abundant ocoyte mitochondfial (I,7o3) DNAo and (I,697) DNA from ~,becontaminating follielecellnuelei, while the two fractions of high buoyant density are identified as (z.7a9) and (z.7z3) rDNA, both containing sequences complementary to ~8-S RNA (Fig. 2a) and xS-S rRNA not shosn~), F6r comparison, somatic (L7~-3) rDIqA from nucleated erythseeytes BDchtm, Btophy~. Acta, x66 (x968) 274--276
:PRELIMINARY NOTES
/~
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Fig, L Analytical certttifugation of the ribosomal satellite DNA from different sources. All samples were centrifuged in a Spinco Model E at z5 °, 44 ooo revdmln for 24 h. The average density of the CsCl was [.7z5 g - c m -a. Exposure series were taken to ensure linear response of the photograph/c record i~ the satellite regions. (a) Isolated r D N A (L7Z 3 g . c m -a) from nucleated erythrocytes together w i t h o,35/~g E. coil DNA (t ,7Io g . c m -B) as a density marker. E. coil DNA also served as z n internal standard for the determination of the rDNA mass in (b) and (v). (b) DNA sample from 9.x.lo~ oocytes. Follicle cells contribute t h e 1.7~ 3 rDNA, while the L7Z9 rDNA is of nucleolar odgin. (c) DNA extracted from 12. i o6 isolated nuclcolL An E. co/i DNA marker has been added. A prominent satellite is found a t a density of 1.7~ 9 g . c m -s. I n b o t h (b) and (c) t h e m~,~s of the nuclear a n d mitochondriat DNA is in large excess to th~.t of t h e r D ~ A. This is because the mitochondrial DNA (3 n g per oocyte) 0 a n d the DNA oi each follicle nucleus (6 p$) exceeds the r D N A mass o£ t h e ooeytc nuelcohis by a factor of 3' xoS and 6oo, respectively. Fig. z. rDIqA complements as revealed b y R N A - D N A hybridization: DlqA f ~ m L8-xo4oocytes or z.o, lo ~ nucleoli wa~ banded ac,~:ording to FL^M~I. B o s e ~.I~vBuRlUSin CsCI (5 ml; !.Tzog- cm -D) in a MSE xo × io mt angle rotor (25~) by centrifuging at 43 ooo rev.]mJn for 35 h. 5-drop fractions were collected in 0, 5 ml o,o! M Tris (pH 8}, the ab~Orbance at z6o m p measured and the DNA el each Iraction denatured w i t h 0. 5 ml i M NaOH. 0.5 mt HCI. o.5 ml r M Tris (pH 8) a n d I mt 3 M ~qaCl were added a n d the DNA qualitatively bound to miltipore HA~VP o.45-p filters according to GILL~SPI~ AND SPIEGELMAN14. The hybridization reaction was carried o u t for 6o m i u at 7o~ in o, 9 M N~t C[-o,o 9 M trisodiumcitrate buyer in t h e presence of (3 #g/ral) aC-l&l~led ~8-S R N A I L I - i o 6 counts/mln per pg). The filters were then washed a n d treated w i t h rihonuclease~L (a} "ooeyt¢" DNA; (b) n u c h o l a r DNA. I n b o t h cases the only DNA capable of hybridizing toribosomal z8-S (and z8-S) rRlgA is the high denstty component (s), while t h e DN& of low buoyant densityis ~baolutelyiner t.No background deduction~ have been applled.Thc distribution of absorbance i s t h a t / o r oocyto DNA. DNA from Microe.oc~gs lysodeikficus served as o. density ma~ker (L73z g.em-~), is also s h o w n (Fig. xa). I t is c l e a r t h a t of t h e t w o r D N A s a t e l l i t e s t h e n u c l e o l a r p r e p a r a t i o n s c o n t a i n o n l y t h e ( L 7 z 9 ) c o m p o n e n t . T h e ( L 7 e 3 ) r D N A also p r e s e n t in th0 "ooeyte" DNA corresponds in density to somatic rDNA and it seems that t h i s D N A m u s t b e c o n t r i b u t e d b y t h e follicle cell nuclei. F r o m F i g , x i t m a y b e c a l c u l a t e d t h a t 9 . x . xo¢ o o c y t e s c o n t a i n a p p r o x , o . z o / ~ g (x,7~9) r D N A o r z z pg~ooeyte. T h i s v a l u e c o m p a r e s v e r y f a v o n r ~ b l y w i t h c y t o c h e m i c a l q u a n t i t a t i o n s of t h e e x t r a D N A (3o 1 ~ ) f o r m e d i n t h e e a r l y o o c y t e n u c l e u s
Biochim. Biop~ys. A¢ta, ~66 (x968) ~74-'~76
276
pI~I~LIMINARYNOTES
in both mutant 1 and wild-type Xenepus is and with values reported independently by B~owN ^~D DAwm n, who used isolated oocyte nuclei, cather thon nuclcoli to determine the amount of amplified rDNA. In three preparations containing x,6 (z.o and LZ)-~O~ nucleoli, 0.I8 (o.~4 and o.o9)P~ (r.7~9) rDNA was recovered, i.e. o.oII pg rDNA per cocyte nucleolus on average. Since there are some x5oo nucleoli in the oocyte nucleus the :full nucleolar complement contributes at least x7 pg r D N A per oocyte, i.e. most, if not all, of the oocyte specific (x.7~9) rDNA is contained in the nucleoli. The nueleolar rDNA of the Xenepus oocyte nucleus is some x4oo-cooo-fold more abundant than the rDNA of a somatic nucleus (o,orz pg)lS. Indeed it is present in greater amount than the calculated 4C mass of the entire oocyte chromosomal DNA (zz pg). Its density corresponds to that reported by GALL2 for the amplified ribosomal eistrons of the oocyte, and is significantly different from that of somatic rDNA. Like somatic rDNA, the rDNA of the ooeyte forms a distinct DNA satellite. Whether it is generated from the somatic rDNA which has an overall lower buoyant density, poses an interesting problem. However, it already seems clear that, contrary to previous theories (of. ref. x), the oocyte rDNA does not arise simply through the copying of the nudcolar organiser DNA as a unit. E~igenetics Research Laboratories, Institute o[ A n i m a l Genetics, University o! Edinburgh, Edinburgh (C/feat Britain)
D. EVANS M . L . BmNSTmL
1 E . I:~RXOWftKA,H, C, ~ACGR1ZOORANDM. L, ~IRNSTIELjggaluve, ~l 7 (~968) 649, z J. O. G^~L, Peon, Natl. Acad. SoL U.S., 60 (x968) .553. 3 M. L. BX~tNS~'~BL,Ann. Roy. Plant Physiol., 18 (~967) zS. 4 M. L. BIRI~S¢IEL.H. W^LLaCE,J. L. SIRLINANDM. FISCHBgRO.Nail. Cancer Ins1. Mo~ograpl~, z3 (x966) 45L 5 M, L, B;RSS~I:gI~,in A, V, S. D~ R~vcg ASP J. KNIGHT,Ciba Foundation Syrup. CellDil/erentiation (I967) 178. 6 M. L. BtRUSXIBL,J, SI~BI~s, J, Puevo~, K. JoN~s, and U, Lo~mNo, Iqaturc, ~x9 (:968) 454. 7 P, B, H~IL~ON, Anal. Chert,, 30 (z958) 9x4 . 8 J. MARUUR,J, Mol. Biol., 3 (z96zl ¢o8. O I. B. DAWID,J. Mol, BioL, I2 {x965}58L 1o A. G. MAcGRBGOR,J. Cell Sci.. in the pre~, II D . D . BROWNAND I. B . DAWID,S~ie~e, I6O (I968) I7I. |2 H. W~,LLACE~ D M, L. B~ssSTml~,Bioehim. BlOphy~. Aeta, xx4 (x966) ~96. 13 W, G. FL~UM, H. E. BOICVa~n H, E, Burn, Bioekim. Btophys. AeI~, xz9 0966) 3~o. 14 D . GILLBBPIEANDS. SPIEGELMAI~,J, ~ol. ~ioL, 1~ (1965) 829,
Received May 17th, 1968 Bioehim. Biophys. Aeta, 166 (x968) 274-276
PKELIMINARY NOTES
PRELIMINARY NOTES BBA 91224 Lo~liw~tion of arnplified ribosomol D N A in the oo~/te of Xenopus I ~ l s There are some z5en nucleoli I in the ooeyte nucleus of Xenopus laevis, the south african clawed toad, compared to one or two in somatic nuclei. The oocyte nuclsoli contain DNA which has arisen by gene amplification (of. ref. z). It hasbeen shown that in somatic coils the nuclcolus is formed largely through the activity of the ribosomal cistrons which are clustered together at the nucleolar organiser (c/. ref. 3). These ribosomal cistroas may be isolated from somatic tissues in a DN'A satellite (rDNA) of a buoyant density of L723 g ' c m -s (refs. 4. 5). In hybridization experiments with pure :DNA it may be demonstrated that 35 % of the satellite mass, and possible more, is made up from ~8-S and x8-S cistruns 0. We have now estimated the mass and buoyant density of the rDNA contained in nncleoli isolated from immature ocoytes of X. laevls. Ovarian tissue from 6-8 Xenopus females was disaggregated by treatment with pronase (aoopg/ml in 0,08 M KCI; 0,02 M ~aCI), The oocytes were fractionatod according to size by floatation ~ in o,o8 M KCI, o,o2 M NaCi and oocytes of overall diameter o,zu-o.45 ram, accompanied by some adhering follicle cells, were collected. This procedure yielded x.to4-2.xo 4 oucytes, The oocytes were suspended in 5 ml of o,4 M sucrose, containing 3 mM Ca¢I2, 0.o8 M KCI and o.oz M NaCI, and ruptured by rapid expulsion through a Pasteur pipette, The homogenate was filtered through three layers of cheese cloth and the nueleolipelleted through ~ M sucrose, 3 mM CaCI¢at zo ooo rev./min for 7 rain in a Spinco rotor SW 25.I at 4°° Light and electron microscopic examination of the preparation shewed the nudceli to be well preserved and free from yolk piatelets. They contained a small fraction of pigment granules and some follicle cell nuclei (<: z %), DNA was extracted according to ~t~ARMUR8, with the modification that after the initiat precipitation with ethar~ol the DIq'A was dissolved in o,25 b[ NaCI, o.oi5 M sodium citrate (pH 7.~), treated with ribonuelease (zoo pg/ml) and exhaustively diaiysed against the solvent at room temperature. The DNA was quantitatively pelleted by centrifugation for 2o h a~: 5o ooo rev./min (I5°) inaSpinco rotor No, 50, Equilibrium centrifugation in CsCl in the analytical centrifuge resolved the nucleolar DNA into two components banding in CsC1 at 1.7z9g'em -s and z.697 g'em -a (Fig, xe). (I,729) DNA was found to be hybridizable to rRNA, hence is identified as rDNA (Fig, zb), The (z.697) DNA which has the same density as somatic (total) nuclear DNA is thought to be derived from the contaminating follicle cell nuclei, each of which contributes some 6 pg DNA9, i,e, some 60o times more D1qA than does each oocyte nueleohis (see below). The DNA extracted from whole oocytes is resolved into three components banding at z,7~,9, z,723 and 1,7ox g,cm-s. The (z.7ox) componentis most likely a composite of the very abundant ocoyte mitochondfial (I,7o3) DNAo and (I,697) DNA from ~,becontaminating follielecellnuelei, while the two fractions of high buoyant density are identified as (z.7a9) and (z.7z3) rDNA, both containing sequences complementary to ~8-S RNA (Fig. 2a) and xS-S rRNA not shosn~), F6r comparison, somatic (L7~-3) rDIqA from nucleated erythseeytes BDchtm, Btophy~. Acta, x66 (x968) 274--276
:PRELIMINARY NOTES
/~
z75
~ ~ i J
~'" I ~ ' ~ /i II i /[ ,
t
I
i
I
/
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e
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Fig, L Analytical certttifugation of the ribosomal satellite DNA from different sources. All samples were centrifuged in a Spinco Model E at z5 °, 44 ooo revdmln for 24 h. The average density of the CsCl was [.7z5 g - c m -a. Exposure series were taken to ensure linear response of the photograph/c record i~ the satellite regions. (a) Isolated r D N A (L7Z 3 g . c m -a) from nucleated erythrocytes together w i t h o,35/~g E. coil DNA (t ,7Io g . c m -B) as a density marker. E. coil DNA also served as z n internal standard for the determination of the rDNA mass in (b) and (v). (b) DNA sample from 9.x.lo~ oocytes. Follicle cells contribute t h e 1.7~ 3 rDNA, while the L7Z9 rDNA is of nucleolar odgin. (c) DNA extracted from 12. i o6 isolated nuclcolL An E. co/i DNA marker has been added. A prominent satellite is found a t a density of 1.7~ 9 g . c m -s. I n b o t h (b) and (c) t h e m~,~s of the nuclear a n d mitochondriat DNA is in large excess to th~.t of t h e r D ~ A. This is because the mitochondrial DNA (3 n g per oocyte) 0 a n d the DNA oi each follicle nucleus (6 p$) exceeds the r D N A mass o£ t h e ooeytc nuelcohis by a factor of 3' xoS and 6oo, respectively. Fig. z. rDIqA complements as revealed b y R N A - D N A hybridization: DlqA f ~ m L8-xo4oocytes or z.o, lo ~ nucleoli wa~ banded ac,~:ording to FL^M~I. B o s e ~.I~vBuRlUSin CsCI (5 ml; !.Tzog- cm -D) in a MSE xo × io mt angle rotor (25~) by centrifuging at 43 ooo rev.]mJn for 35 h. 5-drop fractions were collected in 0, 5 ml o,o! M Tris (pH 8}, the ab~Orbance at z6o m p measured and the DNA el each Iraction denatured w i t h 0. 5 ml i M NaOH. 0.5 mt HCI. o.5 ml r M Tris (pH 8) a n d I mt 3 M ~qaCl were added a n d the DNA qualitatively bound to miltipore HA~VP o.45-p filters according to GILL~SPI~ AND SPIEGELMAN14. The hybridization reaction was carried o u t for 6o m i u at 7o~ in o, 9 M N~t C[-o,o 9 M trisodiumcitrate buyer in t h e presence of (3 #g/ral) aC-l&l~led ~8-S R N A I L I - i o 6 counts/mln per pg). The filters were then washed a n d treated w i t h rihonuclease~L (a} "ooeyt¢" DNA; (b) n u c h o l a r DNA. I n b o t h cases the only DNA capable of hybridizing toribosomal z8-S (and z8-S) rRlgA is the high denstty component (s), while t h e DN& of low buoyant densityis ~baolutelyiner t.No background deduction~ have been applled.Thc distribution of absorbance i s t h a t / o r oocyto DNA. DNA from Microe.oc~gs lysodeikficus served as o. density ma~ker (L73z g.em-~), is also s h o w n (Fig. xa). I t is c l e a r t h a t of t h e t w o r D N A s a t e l l i t e s t h e n u c l e o l a r p r e p a r a t i o n s c o n t a i n o n l y t h e ( L 7 z 9 ) c o m p o n e n t . T h e ( L 7 e 3 ) r D N A also p r e s e n t in th0 "ooeyte" DNA corresponds in density to somatic rDNA and it seems that t h i s D N A m u s t b e c o n t r i b u t e d b y t h e follicle cell nuclei. F r o m F i g , x i t m a y b e c a l c u l a t e d t h a t 9 . x . xo¢ o o c y t e s c o n t a i n a p p r o x , o . z o / ~ g (x,7~9) r D N A o r z z pg~ooeyte. T h i s v a l u e c o m p a r e s v e r y f a v o n r ~ b l y w i t h c y t o c h e m i c a l q u a n t i t a t i o n s of t h e e x t r a D N A (3o 1 ~ ) f o r m e d i n t h e e a r l y o o c y t e n u c l e u s
Biochim. Biop~ys. A¢ta, ~66 (x968) ~74-'~76
276
pI~I~LIMINARYNOTES
in both mutant 1 and wild-type Xenepus is and with values reported independently by B~owN ^~D DAwm n, who used isolated oocyte nuclei, cather thon nuclcoli to determine the amount of amplified rDNA. In three preparations containing x,6 (z.o and LZ)-~O~ nucleoli, 0.I8 (o.~4 and o.o9)P~ (r.7~9) rDNA was recovered, i.e. o.oII pg rDNA per cocyte nucleolus on average. Since there are some x5oo nucleoli in the oocyte nucleus the :full nucleolar complement contributes at least x7 pg r D N A per oocyte, i.e. most, if not all, of the oocyte specific (x.7~9) rDNA is contained in the nucleoli. The nueleolar rDNA of the Xenepus oocyte nucleus is some x4oo-cooo-fold more abundant than the rDNA of a somatic nucleus (o,orz pg)lS. Indeed it is present in greater amount than the calculated 4C mass of the entire oocyte chromosomal DNA (zz pg). Its density corresponds to that reported by GALL2 for the amplified ribosomal eistrons of the oocyte, and is significantly different from that of somatic rDNA. Like somatic rDNA, the rDNA of the ooeyte forms a distinct DNA satellite. Whether it is generated from the somatic rDNA which has an overall lower buoyant density, poses an interesting problem. However, it already seems clear that, contrary to previous theories (of. ref. x), the oocyte rDNA does not arise simply through the copying of the nudcolar organiser DNA as a unit. E~igenetics Research Laboratories, Institute o[ A n i m a l Genetics, University o! Edinburgh, Edinburgh (C/feat Britain)
D. EVANS M . L . BmNSTmL
1 E . I:~RXOWftKA,H, C, ~ACGR1ZOORANDM. L, ~IRNSTIELjggaluve, ~l 7 (~968) 649, z J. O. G^~L, Peon, Natl. Acad. SoL U.S., 60 (x968) .553. 3 M. L. BX~tNS~'~BL,Ann. Roy. Plant Physiol., 18 (~967) zS. 4 M. L. BIRI~S¢IEL.H. W^LLaCE,J. L. SIRLINANDM. FISCHBgRO.Nail. Cancer Ins1. Mo~ograpl~, z3 (x966) 45L 5 M, L, B;RSS~I:gI~,in A, V, S. D~ R~vcg ASP J. KNIGHT,Ciba Foundation Syrup. CellDil/erentiation (I967) 178. 6 M. L. BtRUSXIBL,J, SI~BI~s, J, Puevo~, K. JoN~s, and U, Lo~mNo, Iqaturc, ~x9 (:968) 454. 7 P, B, H~IL~ON, Anal. Chert,, 30 (z958) 9x4 . 8 J. MARUUR,J, Mol. Biol., 3 (z96zl ¢o8. O I. B. DAWID,J. Mol, BioL, I2 {x965}58L 1o A. G. MAcGRBGOR,J. Cell Sci.. in the pre~, II D . D . BROWNAND I. B . DAWID,S~ie~e, I6O (I968) I7I. |2 H. W~,LLACE~ D M, L. B~ssSTml~,Bioehim. BlOphy~. Aeta, xx4 (x966) ~96. 13 W, G. FL~UM, H. E. BOICVa~n H, E, Burn, Bioekim. Btophys. AeI~, xz9 0966) 3~o. 14 D . GILLBBPIEANDS. SPIEGELMAI~,J, ~ol. ~ioL, 1~ (1965) 829,
Received May 17th, 1968 Bioehim. Biophys. Aeta, 166 (x968) 274-276