DEVELOPMENTAL
BIOLOGY
50, 443-456
Mitochondrial
Department
(1976)
Poly(A) RNA Synthesis Sea Urchin Development
of Biology,
during
ROBERT DEVLIN of Vwginin, Chnrlottenuille,
University
Accepted
January
Virginia
Early
22901
27, 1976
The synthesis of mitochondrial messenger RNA during early sea urchin development was examined. OligotdT) chromatography and electrophoresis on aqueous or formamide gels of mitochondrial RNA from pulse-labeled embryos showed the presence of eight distinct poly(A)containing RNA species, ranging in size from 9 to 22 S. Nuclease digestion of these RNAs revealed poly(A) sequences of 4 S size. Using sea urchin anucleate fragments, we were able to demonstrate that all eight messenger RNAs are transcribed from mitochondrial DNA, rather than being transcribed from nuclear DNA and imported into the mitochondria. There was no change in the electrophoretic profile of the eight poly(A) RNAs when embryos were pulsed with [“Hluridine at various times after fertilization. Neither was there any change in the incorporation of 1:‘Hluridine into these species or in the percentage of total newly synthesized mitochondrial RNA that contains poly(A) sequences as development progresses. at a constant rate throughout early Even though these RNAs appear to be transcribed development, they were not detected in mitochondrial polysomes until 18 hr after fertilization.
messenger RNA during early sea urchin development. To what extent these messenger RNAs are transcribed from mitochondrial DNA is presently unclear. Several workers have suggested that some or all of the mitochondrial messenger RNAs are transcribed from nuclear DNA and imported into the mitochondria (see Avadhani et al., 1976, for an excellent review of the topic). In this study, however, we are able to show, using sea urchin anucleate fragments, that all eight of these messenger RNAs are transcribed from mitochondrial DNA.
INTRODUCTION
Recent advances have been made in our understanding of the function of the mitochondrial genome (Avadhani et al., 1976). It is clear that mitochondrial DNA codes for ribosomal and tRNAs used in the mitochondrial protein synthesis system (Dawid, 1972). In this study we examine the synthesis of mitochondrial messenger RNA during early sea urchin development. Previous papers have shown that sea urchin mitochondria are capable of synthesizing high molecular weight RNA (Selvig et al., 1970; Chamberlain, 1970). However, in these studies no evidence was presented which identified this RNA as messenger RNA, and it was suggested that the bulk of the newly synthesized RNA was in fact ribosomal RNA (Chamberlain and Metz, 1972). In this study we have used recently developed techniques for the isolation of messenger RNA to identify eight messenger RNA species in sea urchin mitochondria. We also describe the changes in the electrophoretic pattern and rates of synthesis of mitochondrial Copyright All rights
0 1976 by Academic Press, of reproduction in any form
Inc. reserved
MATERIALS
Handling
AND
of Sea Urchin
METHODS
Eggs
Stronglyocentrotus purpuratus and Lytichinus pictus sea urchins were obtained from Pacific Bio-Marine Co., Venice, Calif. Sea urchins were injected with 2-3 ml of 0.5 M KC1 to induce shedding of gametes. Eggs were collected and washed several times in 200 vol of millipore-filtered artificial sea water (MF-MBL) as prepared by Cavanaugh (1956). Eggs were then fertilized by adding sperm diluted 443
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DEVELOPMENTAL BIOLOGY
l:lO,OOO with MF-MBL. Only batches of eggs that gave more than 95% fertilization were used. After fertilization, embryos were incubated at a concentration of lo5 embryos/ml in MF-MBL which contained 100 units/ml of penicillin and 100 pglml of streptomycin. Embryos were allowed to develop on a gyrotory shaker at 15°C for varying lengths of time. Aseptic conditions were maintained at all times. Incubation
with Isotopes
Embryos were labeled with 20 @X/ml of [5-“Hluridine Ci/mmole), ]2,8(20 “Hladenosine (18 Ci/mmole), or [4,53H]leucine (48 Cilmmole) for varying lengths of time. After incubation, embryos were washed with acidic MF-MBL (MBL in which the pH was adjusted to 4.5 with acetic acid) and then with Ca2+ and MgZ+ free MF-MBL. Isolation of Mitochondrial chondriul Supernatants
and Postmito-
Mitochondria were isolated by suspending embryos in 6 vol of sterile isolation medium (0.25 M sucrose, 0.25 M NaCl, 0.002 M EDTA, 0.03 M Tris, pH 7.8) and homogenizing them with several strokes of a tight-fitting Dounce homogenizer. Nuclei and yolk granules were removed by centrifugation at 2000 g for 10 min in a Sorvall RCB-B centrifuge. Mitochondria were pelleted by centrifugation at 12,000 g for 15 min and washed once with isolation medium. In some experiments, mitochondria were further purified by suspending them in 2 vol of isolation medium, layering them over a 0.6-1.2 M sucrose gradient (in isolation medium), and centrifuging them in a Beckman SW 25.1 rotor at 21,000 rpm for 2 hr. The gradient was fractionated and the mitochondria were located by assaying for cytochrome oxidase activity. When RNA in the cytoplasm but outside the mitochondria (“cytoplasmic RNA”) was examined, embryos were homogenized, mitochondria, nuclei, and yolk were pelleted by centrifugation at 15,000g for 15
VOLUME 50. 1976
min, and RNA was isolated pernatant.
from the su-
RNA Isolation All glassware and solutions used in the isolation procedure were autoclaved, and the solutions were made 0.1% in diethyl pyrocarbonate 30 min before use. Mitochondria were suspended in 1 vol of extraction buffer (1% SDS, 0.1 M NaCl, 0.001 M EDTA, 0.01 M Tris, pH 7.61, and extracted with 2 vol of a 1:l phenol/chloroform mixture for 15 min at 35°C. The organic phase was reextracted with 1 vol of extraction buffer at 65°C. The white interphase material from this second extraction was further extracted with 1 vol of buffer and 2 vol of phenol/chloroform at 65°C. The aqueous phases from the three extractions were pooled and incubated with 500 Fg.g/ml of pronase (Sigma) at 37°C for 30 min, extracted with 2 vol of phenol/chloroform and then with 2 vol of chloroform. The RNA in the final aqueous phase was precipitated with 2.5 vol of ethanol at -20°C. The RNA in the pellet was further purified by suspending it in 2 M lithium chloride (in 0.01 M Tris, pH 7.6) for 1 hr at 4°C. The insoluble high molecular weight RNA was pelleted by centrifugation, dissolved in high salt buffer (0.5% SDS, 0.5 M NaCl, 0.01 M Tris, pH 7.61, heated to 65°C for 5 min, and quick-chilled in ice. The heatdenatured RNA was then warmed to room temperature and passed through a 0.5 x O.l-cm oligo(dT) cellulose column (Collaborative Research, Inc.), which was washed with high salt buffer to remove any RNA not bound to the column. RNA which remained bound to the column [poly(A) containing RNA] was eluted with a low salt buffer (0.5% SDS, 0.01 M Tris, pH 7.6) and made 0.1 M in NaCl. Both the binding and the nonbinding fractions were precipitated with ethanol. Polyacrylamide
Gel Electrophoresis
RNA was analyzed on either 2.5% aqueous or 4% formamide gels. The 2.5%
ROBERT
DEVLIN
Mitochondrial
acrylamide (0.125% bis-acrylamide) gels contained 10% glycerol, 0.4 M Tris, 0.02 M NaH,PO,, 0.002 M EDTA, 0.1% SDS, pH 7.4 (Hirsch and Penman, 1973). Prior to electrophoresis the sample was heated to 65°C for 5 min and quick-chilled. Internal ‘“C 28 and 18 S ribosomal RNA markers from quail myoblasts (a generous gift of Dr. Charles Emerson) were added and the sample subjected to electrophoresis at 3 mA/tube for 6 hr. Formamide gels were prepared according to the procedure of Duesberg and Vogt (1973). The 4% acrylamide (0.25% bisacrylamide) gels were buffered with 0.02 M sodium phosphate, pH 7.4. The RNA samples were dissolved in formamide containing 0.001 M sodium phosphate, pH 7.4, and 10% glycerol. After heat denaturation at 65°C for 5 min, internal laC ribosomal RNA markers were added and the samples were electrophoresed at 5 mA/tube for 6 hr. After electrophoresis, the aqueous and formamide gels were sliced into 1.15-mm sections. Each slice was incubated with 0.5 ml of NCS (Amersham/Searle) at 50°C for 2 hr, and mixed with 10 ml of toluene containing 0.4% PPO and 0.005% POPOP (Beckman). The radioactivity in each sample was then determined with 35% efficiency in a Beckman LS-250 scintillation counter. Polysome Isolation Mitochondrial polysomes were isolated by suspending a mitochondrial pellet in 2 vol of polysome buffer (0.01 M MgC&, 0.05 M NaCl, 0.01 M Tris, pH 7.6) containing 2% Triton. The lysate was incubated at 4°C for 10 min and then centrifuged at 18,000g for 10 min. The supernatant was layered over a 15-30% sucrose gradient (in polysome buffer) and centrifuged in a Beckman SW 41 rotor at 41,000 rpm for 2 hr. The gradient was fractionated, aliquots were taken from each fraction and precipitated with cold 10% TCA, and the precipitate was collected on a Millipore filter.
Poly(A)
RNA
445
Synthesis
Each filter was incubated with 0.4 ml of 0.3 N NaOH for 1 hr; scintillation fluid containing 3% BBS-2 and 7% BBS-3 (Beckman) was added, and the samples were counted to determine the amount of labeled material present in each fraction of the gradient. Cytoplasmic 80 S and mitochondrial 55 S monosomes were run on parallel gradients as markers in each experiment. Material sedimenting faster than 55 S on the gradient was pooled, collected by ethanol precipitation, and chromatographed on an oligo(dT) cellulose column to isolate poly(A) containing RNA. RNA Digestion Poly(A) sequences were isolated by nuclease digestion of RNA that had been extracted with phenol/chloroform and washed with lithium chloride. The RNA was digested with pancreatic and T, RNase according to the procedure of Perlman et al. (1973). The nuclease-resistant RNA was extracted with 2 vol of phenol/ chloroform and then with 2 vol of chloroform. The RNA was precipitated with ethanol and electrophoresed on 10% acrylamide gels. Preparation
of Anucleate
Fragments
L. pictus eggs were washed with acidic MF-MBL to remove membranous material, and then layered over a linear gradient constructed from 1.0 M sucrose and a mixture of equal parts 1.0 M sucrose and MF-MBL (Wilt, 1973). The gradient was centrifuged in a Beckman SW 25.1 rotor at 8000 g for 8 min, and then the speed was increased to 25,000g for 12 min. This procedure splits the eggs into nucleate and anucleate fragments, which band at different positions in the gradient. The anucleate fragments were collected by dripping the gradient through a puncture made in the bottom of the tube, and immediately washed in MF-MBL. The fragments were activated by a twostep process. First, they were incubated for
446
DEVELOPMENTAL BIOLOGY
1 min with lo-” M calcium ionophore A23187 (Eli Lily) and then washed with MF-MBL to remove the ionophore. Next, they were suspended for 20 min in hypertonic MBL (Harvey, 1956) and 1O-2 M ammonium chloride. The activated fragments were washed exhaustively with MF-MBL. RESULTS
Isolation
of RNA
A number of controls were performed to insure that pure poly(A1 RNA was isolated from mitochondria in a quantitative manner. Since harsh conditions were needed to homogenize 21-hr embryos (mesenchyme blastula stage), mitochondria were routinely checked for breakage by assaying the postmitochondrial supernatant for isocitrate dehydrogenase, an enzyme located in the mitochondrial matrix. Less than 6% of the enzyme activity was ever found in the supernatant. To monitor any losses of RNA that occurred during the extraction procedure, “C-ribosomal RNA markers were added to the mitochondrial suspension before RNA extraction in some experiments. At least 97% of the 14CRNA was recovered in every experiment. To be certain that each batch of oligo(dT) cellulose we used could effectively separate poly(A) RNA from non poly(A) containing RNA, we chromatographed a mixture of [“Hlpolyadenylic acid (Miles) and “C-ribosomal RNA. Less than 0.2% of the ribosomal RNA was retained on the column in the presence of high salt buffer and 97.6% of the [“Hlpolyadenylic acid was eluted with low salt buffer. Several workers have shown that there is extensive end labeling of tRNA in sea urchin embryos labeled with [“Hluridine (Gross et al., 1965). Therefore, the isolated RNA was always washed with 2 M lithium chloride to remove low molecular weight RNA. Small RNA molecules (e.g., tRNA) and DNA fragments are soluble in this
VOLUME 50. 1976
solution but high molecular weight RNA is not. When the washed RNA was incubated in 0.3 N NaOH for 18 hr at 37”C, it was rendered 98% soluble in cold TCA. Identification RNA
of Mitochondrial
Poly(A)
To determine if sea urchin mitochondria contain poly(A1 RNA we incubated L. pictus embryos with [“Hluridine for 3 hr and extracted the mitochondrial RNA. It was then chromatographed on an oligo(dT) cellulose column to separate any poly(A) RNA from non poly(A) RNA. To prevent any contamination of the poly(A) RNA by non poly(A1 RNA due to adventitious basepairing between them, the RNA samples were always heat-denatured at 65°C for 5 min before being applied to the column. The bound RNA which was subsequently eluted with low salt buffer was apparently free of non poly(A) RNA since when it was passed through the column a second time, more than 99% of it was retained on the column in the presence of high salt buffer. The poly(A1 RNA and non poly(A) RNA were then analyzed by electrophoresis on 2.5% acrylamide gels (Fig. 1). The poly(A) RNA separated into eight distinct species, while the non poly(A) RNA consisted of two species. These latter two probably correspond to the mitochondrial ribosomal RNAs, since electrophoretic profiles of bulk mitochondrial RNA also exhibit bands coincident with these two species. In many cases the determination of RNA molecular weights on aqueous gels is inaccurate. Therefore, the mitochondrial RNA was electrophoresed on 4% formamide gels (Fig. 2). In such gels the secondary structure of RNA is destroyed and electrophoretic mobility is independent of base composition (Pinder et al., 1972). The poly(A) RNA was resolved into eight species on these gels also. Table 1 lists the apparent molecular weights of these eight RNAs. In order to measure the lengths of mitochondrial poly(A1 sequences, mitochon-
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Mitochondrial
PolylAl
RNA
Synthesis
447
165 & 601
40(
20
20
40 SIICC no
60
80
FIG. 1. Electrophoretic analysis of mitochondrial RNA. L. picks eggs were fertilized and incubated for 3 hr with 20 @G/ml [:‘Hluridine. Mitochondrial RNA was extracted and chromatographed on oligo(dT) cellulose. The RNA fractions were electrophoresed on 2.5% acrylamide gels, sliced, and counted. (A) Poly(A) RNA; (B) non poly(A) RNA.
drial RNA was extracted from embryos which had been labeled with CZsH]adenosine for 3 hr. After nuclease digestion of the extracted RNA, the resistant fragments were electrophoresed on 10% acrylamide gels. The fragments migrated as a broad peak having a modal value of 4 S (Fig. 3). This represents about 60-70 nucleotides and is in agreement with similar determinations using mitochondria from other sources (Perlman et al., 1973; Avadhani et al., 1973a). Using similar procedures we find that poly(A) sequences of cytoplasmic RNA of sea urchins migrate as a 7 S peak. Isolation
of Mitochondrial
Polysomes
Mitochondrial polysomes were prepared from embryos labeled with [:‘H]leucine in the presence of emetine, a specific inhibitor of nonmitochondrial protein synthesis. The polysomal profile (Fig. 4) exhibits a broad distribution of rapidly sedimenting labeled material, with a large peak at 55 S
and smaller ones at about 110 and 160 S. These probably represent the mitochondrial monomer, dimer, and trimer regions of the gradient. When chloramphenicol, a specific inhibitor of mitochondrial protein synthesis, was added 10 min before the [“Hlleucine pulse there were essentially no counts in the rapidly sedimenting material. Therefore, the labeled material in these structures is the result of actively translating mitochondrial polysomes, and is not due to cytoplasmic protein synthesis or to nonspecific binding of [“Hlleucine. To further demonstrate that the rapidly sedimenting labeled structures are indeed mitochondrial polysomes, we incubated embryos with 150 pg/ml of puromycin 5 min before homogenization. Puromycin will release nascent chains from the polysomes. The data in Fig. 4 indicate that 91% of the labeled material in the structures sedimenting faster than 55 S is absent, indicating that these structures are in fact mitochondrial polysomes.
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DEVELOPMENTAL BIOLOGY
VOLUME 50, 1976 TABLE MOLECULAR
WEIGHTS PoLYCA)
Component
1 OF MITOCHONDRIAL
RNAs Molecular weight” x 10-S 5.6 5.1 4.6 4.2 4.0 3.6 3.4 3.1
LI From Fig. 2(B).
2ocx 50
70
90
Slice no
110
130
FIG. 2. Electrophoretic analysis of mitochondrial RNA on formamide gels. (A) The RNA sample was dissolved in a solution of formamide buffered with 0.001 M sodium phosphate, pH 7.1, and heated to 65°C before being applied to a l&cm gel. (B) The positions of the eight components on the gel are indicated by arrows on the standard curve. This curve was constructed from the electrophoretic mobilities and molecular weights of the large and small mitochondrial ribosomal RNAs. These molecular weights were taken as 5.4 x lo” and 3.4 x 10” in accordance with published values of other animal mitochondrial ribosomal RNAs (e.g., Ojala and Attardi, 1974; Dawid and Chase, 1972).
c
E ci ; ,I 1coc
c
To determine if any of the eight newly synthesized poly(A) RNA species is present in the polysomes, embryos were incubated with [“Hluridine and the mitochondrial polysomes were prepared. The polysomal profile is shown in Fig. 5; the material sedimenting faster than 55 S was collected, made 1% in SDS and 0.5 M in NaCl, chromatographed on an oligo(dT) cellulose column to isolate poly(A) RNA, and electrophoresed on 2.5% gels. The electrophoretic profile (Fig. 6) shows that all eight poly(A) RNA species seen in the mitochondria are also present in the polysomes.
20
40 SIICC no
60
80
FIG. 3. Poly(A) sequences isolated from mitochondrial and cytoplasmic poly(A) RNA. 5’. parpuratus eggs were fertilized and incubated for 3 hr with 20 $.Xml of [:‘Hladenosine. Mitochondrial and cytoplasmic poly(A) RNA was prepared and digested with nucleases. The nuclease resistant fragments were electrophoresed on 10% acrylamide gels, sliced, and counted. 14C 5 and 4 S markers were added to each sample before electrophoresis. O-O, Mitochondrial poly(A) sequences; O-O, cytoplasmic poly(A) sequences.
Anucleate
Fragments
In order to determine the genetic origin (mitochondrial or nuclear) of the poly(A) RNAs found in the mitochondria, sea ur-
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Poly(A)
10 Fraction
15
RNA
Synthesis
449
20
no
FIG. 4. Mitochondrial polysomal profile of [:‘Hlleucine-labeled embryos. L. picks 18-hr embryos were incubated with 10m4M emetine for 15 min and then 20 pCi/ml of [:‘Hlleucine was added and the embryos incubated for 30 min. Mitochondrial polysomes were analyzed by sucrose gradient centrifugation. O-0, Control polysomes; O--O, polysomes isolated from embryos to which 150 pg/ml of puromycin was added 5 min prior to embryo homogenization; x-x, polysomes isolated from embryos to which 50 pg/ml of chloramphenicol was added 15 min before the [“Hlleucine.
150
50
FIG. 5. Mitochondrial polysomal profile of [:lH]uridine-labeled embryos. L. pi&us 18-hr embryos were labeled with 20 @.X/ml of [:‘Hluridine for 2 hr. The mitochondrial polysomes were then prepared by sucrose gradient centrifugation.
FIG. 6. Poly(A) RNA from mitochondrial polysomes. The material sedimenting faster than 55 S in Fig. 5 (the area enclosed in the bracket) was collected, chromatographed on oligo(dT) cellulose, and the poly(A) RNA was electrophoresed on 2.5% gels.
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DEVELOPMENTAL BIOLOGY
chin anucleate fragments were analyzed. Sea urchin eggs can be split into nucleate and anucleate fragments, by centrifugation in a sucrose gradient. The anucleate fragments, which contain many of the egg’s mitochondria, can be activated artificially and will cleave several times. During this time the mitochondria synthesize high molecular weight RNA (Chamberlain, 1970; Selvig et al., 1970). This presents an excellent opportunity to study mitochondrial RNA synthesis in the absence of nuclear RNA synthesis. Extensive microscopic examination of the anucleate fragments revealed no contamination from either whole eggs or nucleate fragments. Activated anucleate fragments of L. pictus eggs were incubated with 13Hluridine for 3 hr, and the mitochondrial poly(A) was extracted and electrophoresed on 2.5% acrylamide gels. The electrophoretic profile (Fig. 7) consists of eight peaks of the same size and overall pattern as mitochondrial poly(A) RNA from whole embryos. This demonstrates conclusively that the eight poly(A) RNA species are all transcribed from mitochondrial DNA, not nuclear DNA. Poly(A)
RNA during
Development
In order to detect any changes in the number or amount of poly(A) RNA synthesized during early sea urchin development, mitochondrial RNA was extracted from S. purpuratus embryos pulsed with VH]uridine at various times after fertilization. The RNA was chromatographed on oligo(dT) cellulose and the electrophoretic profiles of the RNA from the various stages of development were compared in Fig. 8. The electrophoretic mobilities of the eight S. purpuratus poly(A) RNAs are slightly larger than the corresponding poly(A) RNAs from L. picks, though the overall pattern is very similar. The mobilities of the non poly(A) RNAs in the two species are identical. As can be seen in Fig. 8, the profiles of the eight RNA spe-
VOLUME 50, 1976
0
1
20
FIG. 7. Poly(A) L. pictus anucleate incubated for 3 hr Poly(A) RNA was electrophoresed on
40 Si1ce no
60
80
RNA from anucleate fragments. fragments were activated and with 20 pCi/ml of [:‘Hluridine. prepared from mitochondria and 2.5% gels.
ties are identical at each of the times studied. Thus there appears to be no qualitative changes in the pattern of mitochondrial RNA synthesis during early sea urchin development. There is, however, a progressive increase in the amount of material larger than peak 1 synthesized as development proceeds. Since it seemed likely that this material represents cytoplasmic contamination of mitochondrial RNA, several controls were run. First, ethidium bromide (a specific inhibitor of mitochondrial RNA and protein synthesis) was added to the embryos 15 min before the [:%Hluridine pulse. The results, shown in Fig. 8, indicate that while the synthesis of RNA larger than peak 1 is only slightly inhibited, the synthesis of RNA smaller than peak 1 is completely inhibited by this compound. Table 2 shows the sensitivity of mitochondrial and cytoplasmic RNA synthesis to ethidium bromide at 3 and 21 hr after fertilization. It is evident that mito-
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Mitochondrial
Poly(A)
RNA
451
Synthesis
i
d
28s J
185 I
o-
o-
'0 -
0
r
a
I
C
2occ-
3000-
J 2000.
lOOO-
1000 -
if 0
, 20
10
GO
80
FIG. 8. Mitochondrial RNA from different stages of development. S. purpuratus embryos were pulsed for 3 hr with 20 &i/ml of [“Hluridine at 0, 8, and 18 hr after fertilization. The mitochondrial poly(A) RNA and non poly(A) RNA was prepared and electrophoresed on 2.5% gels. (A) Mitochondrial poly(A) RNA from 3-hr embryos; (B) mitochondrial poly(A) RNA from 11-hr embryos; (C) mitochondrial poly(A) RNA from 21-hr embryos; (D) mitochondrial non poly(A) RNA from 21-hr embryos [the mitochondrial non poly(A) RNA from 3-hr and 11-hr embryos is not shown but is exactly the same as that shown for the 21-hr embryos]. O--O, Control poly(A) RNA; O--O, poly(A) RNA from embryos to which 25 pg/ml of ethidium bromide was added 30 min before the [:‘Hluridine pulse.
452
DEVELOPMENTAL BIOLOGY
185 4.
(
_ 20
40 SllCf
no
60
80
FIG. 9. Electrophoretic analysis of cytoplasmic poly(A) RNA. S. purpuratus 18-hr embryos were labeled for 3 hr with 20 &i/ml of [:‘H]uridine. RNA was extracted from the postmitochondrial supernatant, chromatographed on oligo(dT) cellulose, and the poly(A) RNA electrophoresed on 2.5% gels.
chondrial RNA synthesis, particularly mitochondrial poly(A) RNA synthesis, is much more sensitive to the drug than cytoplasmic RNA synthesis. Next, the electrophoretic profile of cytoplasmic poly(A) RNA synthesized 21 hr after fertilization was examined. The pattern, shown in Fig. 9, indicates that 98% of this RNA is larger than peak 1. If only 0.1% of this cytoplasmic poly(A) RNA were to contaminate the mitochondria, it would account for the labeled material larger than peak 1 found in the mitochondrial RNA profile at the mesenchyme blastula stage. Finally, if mitochondria are purified by sucrose gradient centrifugation prior to RNA extraction, the material larger than peak 1 is greatly reduced, though not eliminated. The recovery of RNA species smaller than peak 1, however, is completely unaffected. The data from these
VOLUME 50, 1976
three controls indicate that the material larger than peak 1 in the profiles from mitochondria at the later developmental stages is indeed cytoplasmic contamination. The relative amount of newly synthesized mitochondrial RNA which contains poly(A) sequences was also determined at various times during development (Table 3). There is no significant change in the percentage of mitochondrial poly(A) RNA synthesized in stages up to mesenchyme blastula. This is in contrast to the percentage of cytoplasmic poly(A) RNA synthesized, which changes from 13 to 39% as development proceeds. The relative incorporation of [“Hluridine into mitochondrial and cytoplasmic poly(A) RNA at various times during development was also studied (Table 4). The relative rate of incorporation of [“Hluridine into mitochondrial poly(A) RNA remains essentially constant at all times studied, but the incorporation into cytoplasmic poly(A) RNA increases more than loo-fold during development. The rate of [“Hluridine incorporation into mesenchyme blastula mitochondrial poly(A) RNA is slightly larger than at earlier times, but this higher rate is due to contamination by cytoplasmic RNA. If only the poly(A) RNA smaller than peak 1 is counted, the incorporation at this stage is 41,000 cpm/106 embryos. There is no change in the electrophoretic profile of mitochondrial poly(A) RNA or its apparent rate of synthesis during early sea urchin development. However, there is an increase in the amount of labeled RNA found in the mitochondrial polysomes as development proceeds. Figure 10 shows the mitochondrial polysomal profiles prepared from pulse-labeled embryos at 3, 11, and 21 hr after fertilization. Between 11 and 21 hr of development there is a sixfold increase in the labeled material sedimenting faster than 55 S. If this material is chromatographed on collected and oligo(dT) cellulose to isolate poly(A) RNA
ROBERT
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Mitochondrial
Poly(Ai
TABLE SENSITIVITY
OF MITOCHONDRIAL
RNA
2
AND CYTOPLASMIC
RNA SYNTHESIS
Percentage
TO ETHIDIUM
Total RNA RNA
96 38
Poly(A) 99 36
BROMIDE”
RNA inhibition
3-Hr embryos
Mitochondrial Cytoplasmic
453
Synthesis
21-Hr embryos Non poly(A1 92 47
Total
Poly(A1
Non poly(A1
80 13
68 20
74 16
fl At 0 and 18 hr after fertilization 10” S. purpuratus embryos were split into two groups, one of which was incubated for 30 min with 25 Fg/ml of ethidium bromide. Then all embryos were pulsed with 20 &i/ml [“Hluridine for 3 hr. RNA from mitochondria and postmitochondrial supernatants was extracted and washed with lithium chloride. An aliquot from each group was precipitated with cold 10% TCA, filtered, and counted to determine the radioactivity in total mitochondrial and cytoplasmic RNA. The remainder was chromatographed on oligo(dT) cellulose, and aliquots of the poly(A) RNA and non poly(A) RNA were precipitated with TCA and counted.
TABLE
TABLE
3
CHANGES IN THE PERCENTAGE OF MITOCHONDRIAL AND CYTOPLASMIC RNA CONTAINING PoLY(A) SEQUENCES AT DIFFERENT STAGES OF DEVELOPMENT”
3-Hr embryos (%‘o) Mitochondrial Cytoplasmic
11-Hr embryos (70)
53 13
50 20
Radioactivity of poly(A) (cpm/lO” embryos)
21-Hr embryos (o/o) 49 39
* S. purpuratus embryos were labeled for 3 hr with 20 @/ml of [:‘Hluridine. Mitochondrial and cytoplasmic poly(A1 RNA was extracted, washed with lithium chloride, and chromatographed on oligo(dT) cellulose to determine the percentage of total labeled RNA which contains poly(A1 sequences.
there is a corresponding sixfold increase in the amount of labeled poly(A) RNA found in the polysomes. DISCUSSION
In the present study we have demonstrated for the first time the presence of poly(A) containing RNA in mitochondria of sea urchin embryos. This RNA has a poly(A) sequence of 4 S size compared to a 7 S size poly(A) sequence found on cytoplasmic sea urchin poly(A) RNA. In view of the relationship between poly(A) sequences and messenger RNA, and because of the presence of the mitochondrial poly(A) RNA in the polysomes, this RNA almost certainly represents mitochondrial messenger RNA.
4
RELATIVE INCORPORATION OF [“HIURIDINE INTO MITOCHONDRIAL AND CYTOPLASMIC PoLY(A) RNA AT DIFFERENT STAGES OF DEVELOPMENT”
RNA
3-Hr embryos
11-Hr embryos
Mitochondrial (cpm) Cytoplasmic (cm)
42,000
40,000
58,000
12,000
650,000
1,400,000
u S. purpuratus with 20 pCi/ml of cytoplasmic poly(A) phoresed as in Fig. totaled to determine
embryos were labeled for 3 hr [:‘H]uridine. Mitochondrial and RNA was extracted and electro1. The counts on each gel were the relative incorporation rate.
21-Hr embryos
It is unlikely that any of the eight poly(A) RNA species is a degradation product of a larger species, because the electrophoretic profile is very reproducible and there is no evidence of degradation of “C-ribosomal RNA markers added before RNA extraction. It is also unlikely that any of these eight species results from aggregation of smaller RNAs, since the electrophoretic profile observed on aqueous gels is also displayed on gels run in the presence of formamide, which prevents the aggregation of RNA. The possibility that there is a precursor-product relationship between some of the poly(A) RNA species cannot be ruled out since kinetic studies were not done; but since all eight species
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DEVELOPMENTAL BIOLOGY
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5
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VOLUME 50, 1976
15
20
Fractionno
FIG. 10. Mitochondrial polysomal profiles from different stages of development. L. pictus embryos were incubated for 2 hr with 20 &i/ml of [“Hluridine at 0, 8, and 18 hr after fertilization. Polysomes were prepared from the mitochondria by sucrose gradient centrifugation for 3 hr. x-x, Polysomes from 2-hr polysomes from 20-hr embryos. embryos; O-O, polysomes from lo-hr embryos; O---O,
are present in the polysomes, this possibility is unlikely. Therefore, these poly(A) RNAs appear to represent eight unique mitochondrial messenger RNAs. Genetic Origin
of the Poly(A)
RNA
One of the most controversial aspects of mitochondrial biogenesis concerns the genetic origin of mitochondrial messenger RNA. Several lines of evidence suggest that at least some messenger RNAs may be transcribed from nuclear DNA and imported into the mitochondria. The small size of the mitochondrial genome in animal cells has prompted the suggestion that mitochondria have insufficient genetic information to account for the known mitochondrially made proteins and ribosomal RNA (Dawid, 1972). It has been shown that isolated mitochondria are capable of taking up and translating many kinds of RNAs (Swanson, 1972; Kiselev and Gaitskhoki, 1972; Dimitriadis and Georgatos, 1974). It has also been proposed that all of the poly(A) RNA in mitochondria of Ascites cells is of nuclear origin (Avadhani et al., 1973b).
However, our results show that there is no importation of nuclear RNA into mitochondria during early sea urchin development. If any of the eight newly synthesized poly(A) RNA species were synthesized in the nucleus and imported into the mitochondria, it would not appear in the electrophoretic profiles of poly(A) RNA made in anucleate fragments. Because the electrophoretic profile of 13Hluridine-labeled mitochondrial poly(A) RNA from anucleate fragments is identical to the pattern obtained from whole embryos, we feel this constitutes conclusive proof that newly synthesized nuclear RNA is not imported into sea urchin mitochondria during early sea urchin development. Size of the Poly(A)
RNA
Assuming that the molecular weight of the 4.45pm L. pictus mitochondrial DNA molecule (Piko et al., 1968) is 9.5 million daltons, the sum of the eight poly(A) RNA species, the large and small ribosomal RNAs, and the 15 known tRNAs account for 48% of the mitochondrial genome. If the equivalent of only one strand of mito-
ROBERT DEVLIN
Mitochondrial
chondrial DNA is transcribed into functional information, and there is only one copy of each of the poly(A) RNAs in the genome, the RNAs listed above comprise 96% of the functional sequences present on mitochondrial DNA. This would appear to leave no room for any other messenger RNAs or extensive spacer regions on the mitochondrial DNA. Poly(A)
RNA during
Development
Our results indicate there is no change in the electrophoretic profile of newly synthesized mitochondrial poly(A) RNA or non poly(A) RNA during early sea urchin development. Also, there is no change in the percentage of mitochondrial RNA which contains poly(A) sequences as development progresses. Nor is there any apparent change in the relative rate of incorporation of [3H]uridine into mitochondrial poly(A) RNA during early development, although pool sizes were not measured. These findings suggest that virtually the entire functional mitochondrial genome is transcribed at a constant rate throughout early development. Since newly synthesized poly(A) RNA does not appear to reach the polysomes for several hours after fertilization, it appears that translational control mechanisms may be more important in mitochondrial biogenesis during early development than transcriptional mechanisms. It would also indicate that mitochondrial transcription and translation are not coupled, as has been suggested (Chooi and Laird, 1974). I express sincere thanks to Dr. Ronald F. Swanson for his continued support and encouragement. I am also endebted to Dr. Charles Emerson for his numerous helpful suggestions and advice, and to Dr. Irwin R. Konigsberg for his careful editing of this manuscript. This work was supported in part by a Training Program in Developmental Biology (HD 00430) from the National Institutes of Health to the University of Virginia. REFERENCES AVADHANI, N. G., KUAN, P., VANDER LEN, P., and RUTMAN, R. (1973a). Polyadenylic acid sequences
Poly(AA) RNA
Synthesis
455
in mitochondrial RNA. Biochem. Biophys. Res. Commun. 51, 1090-1096. AVADHANI, N. G., BATTULA, N., and RUTMAN, R. (1973b). Messenger RNA metabolism in mammalian mitochondria. Origin of ethidium bromide resistent polyadenylic acid containing RNA in Ehrlich Ascites mitochondria. Biochemistry 12, 4123-4128. AVADHANI, N. G., LEWIS, F., and RUTMAN, R. (1976). Mitochondrial RNA and protein metabolism. Subcellular Biochem., in press. CAVANAUGH, G. M. (19561. In “Formulae and Methods,” p. 55. Woods Hole, Mass. CHAMBERLAIN, J. P. (19701. RNA synthesis in anucleate egg fragments and normal embryos of the sea urchin Arbacia punctulata. Biochim. Biophys. Acta 212, 1833193. CHAMBERLAIN, J. P., and METZ, C. B. (1972). Mitochondrial RNA synthesis in sea urchin embryos. J. Mol. Biol. 64, 593-607. CHOOI, W. Y., and LAIRD, C. D. (1974). Visualization of transcription and translation in mitochondria of Drosophila melanogaster. J. Cell Biol. 63, 60a. DAWID, I. B. (1972). Mitochondrial RNA in Xenopus laeuis. I. The expression of the mitochondrial genome. J. Mol. Biol. 63, 201-216. DAWID, I. B., and CHASE, J. W. (1972). Mitochondrial RNA in Xenopus laeuis. II. Molecular weights and other physical properties of mitochondrial ribosomal and 4S RNA. J. Mol. Biol. 63, 217-231. DIMITRIADIS, G., and GEORGATSOS,J. (1974). Induction of protein synthesis in mitochondria by exogenous RNA. Synthesis of rabbit globin by isolated mitochondria of Tetrahymena pyriformis. FEBS Lett. 46, 96-102. DUESBERG, P., and VOGT, P. (1973). Gel electrophoresis of avian Leukosis and sarcoma viral RNA in formamide: Comparison with other viral and cellular RNA species. J. Vir. 12, 594-599. GROSS, P. R., KRAEMER, K., and MALKIN, L. I. (1965). Base composition of RNA synthesized during cleavage of the sea urchin embryo. Biochem. Biophys. Res. Commun. 18, 569-575. HARVEY, E. B. (1956). In “The American Arbacia and Other Sea Urchins.” Princeton University Press, Princeton, New Jersey. HIRSCH, M., and PENMAN, S. (1973). Mitochondrial polyadenylic acid-containing RNA: Localization and characterization. J. Mol. Biol. 80, 379-391. HIRSCH, M., and PENMAN, S. (1974). The messengerlike properties of the poly(Al+ RNA in mammalian mitochondria. Cell 3, 335-339. KISELEV, O., GAITSKHOKI, V., and NEIFAKH, F. (1975). On the transfer of nuclear RNA into isolated mitochondria. Further evidence for template properties of nuclear RNA taken up by isolated mitochondria. Mol. Cell. Biochem. 6, 149-153. OJALA, D., and ATTARDI, G. (19741. Identification
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DEVELOPMENTAL BIOLOGY
and partial characterization of multiple discrete polyadenylic acid containing RNA components coded for by He La cell mitochondrial DNA. J. Mol. Biol. 88, 205-219. PERLMAN, S., ABELSON, H. T., and PENMAN, S. (1973). Mitochondrial protein synthesis: RNA with the properties of eukaryotic messenger RNA. Proc. Nat. Acad. Sci USA 70, 350-353. PIKO, L., BLAIR, D. G., TYLER, A., and VINOGRAD, J. (1968). Cytoplasmic DNA in the unfertilized sea urchin egg: Physical properties of circular mitochondrial DNA and the occurrence of catenated forms. Proc. Nat. Acad. Sci. USA 59, 838-845.
VOLUME 50, 1976
PINDER, J. C., STAYNOV, D. Z., and GRATZER, W. B. (19741. Electrophoresis of RNA in formamide. Biochemistry 13, 5373-5377. SELVIG, S. E., GROSS, P. R., and HUNTER, A. L. (19701. Cytoplasmic synthesis of RNA in the sea urchin embryo. Develop. Biol. 22, 343-365. SWANSON, R. F. (1971). Incorporation ofhigh molecular weight polynucleotides by isolated mitochondria. Nature (London) 231, 31-34. WILT, F. H. (1973). Polyadenylation of maternal RNA of sea urchin eggs after fertilization. Proc. Nat. Acad. Sci. USA 70, 2345-2349.
BIOLOGY
50, 443-456
Mitochondrial
Department
(1976)
Poly(A) RNA Synthesis Sea Urchin Development
of Biology,
during
ROBERT DEVLIN of Vwginin, Chnrlottenuille,
University
Accepted
January
Virginia
Early
22901
27, 1976
The synthesis of mitochondrial messenger RNA during early sea urchin development was examined. OligotdT) chromatography and electrophoresis on aqueous or formamide gels of mitochondrial RNA from pulse-labeled embryos showed the presence of eight distinct poly(A)containing RNA species, ranging in size from 9 to 22 S. Nuclease digestion of these RNAs revealed poly(A) sequences of 4 S size. Using sea urchin anucleate fragments, we were able to demonstrate that all eight messenger RNAs are transcribed from mitochondrial DNA, rather than being transcribed from nuclear DNA and imported into the mitochondria. There was no change in the electrophoretic profile of the eight poly(A) RNAs when embryos were pulsed with [“Hluridine at various times after fertilization. Neither was there any change in the incorporation of 1:‘Hluridine into these species or in the percentage of total newly synthesized mitochondrial RNA that contains poly(A) sequences as development progresses. at a constant rate throughout early Even though these RNAs appear to be transcribed development, they were not detected in mitochondrial polysomes until 18 hr after fertilization.
messenger RNA during early sea urchin development. To what extent these messenger RNAs are transcribed from mitochondrial DNA is presently unclear. Several workers have suggested that some or all of the mitochondrial messenger RNAs are transcribed from nuclear DNA and imported into the mitochondria (see Avadhani et al., 1976, for an excellent review of the topic). In this study, however, we are able to show, using sea urchin anucleate fragments, that all eight of these messenger RNAs are transcribed from mitochondrial DNA.
INTRODUCTION
Recent advances have been made in our understanding of the function of the mitochondrial genome (Avadhani et al., 1976). It is clear that mitochondrial DNA codes for ribosomal and tRNAs used in the mitochondrial protein synthesis system (Dawid, 1972). In this study we examine the synthesis of mitochondrial messenger RNA during early sea urchin development. Previous papers have shown that sea urchin mitochondria are capable of synthesizing high molecular weight RNA (Selvig et al., 1970; Chamberlain, 1970). However, in these studies no evidence was presented which identified this RNA as messenger RNA, and it was suggested that the bulk of the newly synthesized RNA was in fact ribosomal RNA (Chamberlain and Metz, 1972). In this study we have used recently developed techniques for the isolation of messenger RNA to identify eight messenger RNA species in sea urchin mitochondria. We also describe the changes in the electrophoretic pattern and rates of synthesis of mitochondrial Copyright All rights
0 1976 by Academic Press, of reproduction in any form
Inc. reserved
MATERIALS
Handling
AND
of Sea Urchin
METHODS
Eggs
Stronglyocentrotus purpuratus and Lytichinus pictus sea urchins were obtained from Pacific Bio-Marine Co., Venice, Calif. Sea urchins were injected with 2-3 ml of 0.5 M KC1 to induce shedding of gametes. Eggs were collected and washed several times in 200 vol of millipore-filtered artificial sea water (MF-MBL) as prepared by Cavanaugh (1956). Eggs were then fertilized by adding sperm diluted 443
444
DEVELOPMENTAL BIOLOGY
l:lO,OOO with MF-MBL. Only batches of eggs that gave more than 95% fertilization were used. After fertilization, embryos were incubated at a concentration of lo5 embryos/ml in MF-MBL which contained 100 units/ml of penicillin and 100 pglml of streptomycin. Embryos were allowed to develop on a gyrotory shaker at 15°C for varying lengths of time. Aseptic conditions were maintained at all times. Incubation
with Isotopes
Embryos were labeled with 20 @X/ml of [5-“Hluridine Ci/mmole), ]2,8(20 “Hladenosine (18 Ci/mmole), or [4,53H]leucine (48 Cilmmole) for varying lengths of time. After incubation, embryos were washed with acidic MF-MBL (MBL in which the pH was adjusted to 4.5 with acetic acid) and then with Ca2+ and MgZ+ free MF-MBL. Isolation of Mitochondrial chondriul Supernatants
and Postmito-
Mitochondria were isolated by suspending embryos in 6 vol of sterile isolation medium (0.25 M sucrose, 0.25 M NaCl, 0.002 M EDTA, 0.03 M Tris, pH 7.8) and homogenizing them with several strokes of a tight-fitting Dounce homogenizer. Nuclei and yolk granules were removed by centrifugation at 2000 g for 10 min in a Sorvall RCB-B centrifuge. Mitochondria were pelleted by centrifugation at 12,000 g for 15 min and washed once with isolation medium. In some experiments, mitochondria were further purified by suspending them in 2 vol of isolation medium, layering them over a 0.6-1.2 M sucrose gradient (in isolation medium), and centrifuging them in a Beckman SW 25.1 rotor at 21,000 rpm for 2 hr. The gradient was fractionated and the mitochondria were located by assaying for cytochrome oxidase activity. When RNA in the cytoplasm but outside the mitochondria (“cytoplasmic RNA”) was examined, embryos were homogenized, mitochondria, nuclei, and yolk were pelleted by centrifugation at 15,000g for 15
VOLUME 50. 1976
min, and RNA was isolated pernatant.
from the su-
RNA Isolation All glassware and solutions used in the isolation procedure were autoclaved, and the solutions were made 0.1% in diethyl pyrocarbonate 30 min before use. Mitochondria were suspended in 1 vol of extraction buffer (1% SDS, 0.1 M NaCl, 0.001 M EDTA, 0.01 M Tris, pH 7.61, and extracted with 2 vol of a 1:l phenol/chloroform mixture for 15 min at 35°C. The organic phase was reextracted with 1 vol of extraction buffer at 65°C. The white interphase material from this second extraction was further extracted with 1 vol of buffer and 2 vol of phenol/chloroform at 65°C. The aqueous phases from the three extractions were pooled and incubated with 500 Fg.g/ml of pronase (Sigma) at 37°C for 30 min, extracted with 2 vol of phenol/chloroform and then with 2 vol of chloroform. The RNA in the final aqueous phase was precipitated with 2.5 vol of ethanol at -20°C. The RNA in the pellet was further purified by suspending it in 2 M lithium chloride (in 0.01 M Tris, pH 7.6) for 1 hr at 4°C. The insoluble high molecular weight RNA was pelleted by centrifugation, dissolved in high salt buffer (0.5% SDS, 0.5 M NaCl, 0.01 M Tris, pH 7.61, heated to 65°C for 5 min, and quick-chilled in ice. The heatdenatured RNA was then warmed to room temperature and passed through a 0.5 x O.l-cm oligo(dT) cellulose column (Collaborative Research, Inc.), which was washed with high salt buffer to remove any RNA not bound to the column. RNA which remained bound to the column [poly(A) containing RNA] was eluted with a low salt buffer (0.5% SDS, 0.01 M Tris, pH 7.6) and made 0.1 M in NaCl. Both the binding and the nonbinding fractions were precipitated with ethanol. Polyacrylamide
Gel Electrophoresis
RNA was analyzed on either 2.5% aqueous or 4% formamide gels. The 2.5%
ROBERT
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Mitochondrial
acrylamide (0.125% bis-acrylamide) gels contained 10% glycerol, 0.4 M Tris, 0.02 M NaH,PO,, 0.002 M EDTA, 0.1% SDS, pH 7.4 (Hirsch and Penman, 1973). Prior to electrophoresis the sample was heated to 65°C for 5 min and quick-chilled. Internal ‘“C 28 and 18 S ribosomal RNA markers from quail myoblasts (a generous gift of Dr. Charles Emerson) were added and the sample subjected to electrophoresis at 3 mA/tube for 6 hr. Formamide gels were prepared according to the procedure of Duesberg and Vogt (1973). The 4% acrylamide (0.25% bisacrylamide) gels were buffered with 0.02 M sodium phosphate, pH 7.4. The RNA samples were dissolved in formamide containing 0.001 M sodium phosphate, pH 7.4, and 10% glycerol. After heat denaturation at 65°C for 5 min, internal laC ribosomal RNA markers were added and the samples were electrophoresed at 5 mA/tube for 6 hr. After electrophoresis, the aqueous and formamide gels were sliced into 1.15-mm sections. Each slice was incubated with 0.5 ml of NCS (Amersham/Searle) at 50°C for 2 hr, and mixed with 10 ml of toluene containing 0.4% PPO and 0.005% POPOP (Beckman). The radioactivity in each sample was then determined with 35% efficiency in a Beckman LS-250 scintillation counter. Polysome Isolation Mitochondrial polysomes were isolated by suspending a mitochondrial pellet in 2 vol of polysome buffer (0.01 M MgC&, 0.05 M NaCl, 0.01 M Tris, pH 7.6) containing 2% Triton. The lysate was incubated at 4°C for 10 min and then centrifuged at 18,000g for 10 min. The supernatant was layered over a 15-30% sucrose gradient (in polysome buffer) and centrifuged in a Beckman SW 41 rotor at 41,000 rpm for 2 hr. The gradient was fractionated, aliquots were taken from each fraction and precipitated with cold 10% TCA, and the precipitate was collected on a Millipore filter.
Poly(A)
RNA
445
Synthesis
Each filter was incubated with 0.4 ml of 0.3 N NaOH for 1 hr; scintillation fluid containing 3% BBS-2 and 7% BBS-3 (Beckman) was added, and the samples were counted to determine the amount of labeled material present in each fraction of the gradient. Cytoplasmic 80 S and mitochondrial 55 S monosomes were run on parallel gradients as markers in each experiment. Material sedimenting faster than 55 S on the gradient was pooled, collected by ethanol precipitation, and chromatographed on an oligo(dT) cellulose column to isolate poly(A) containing RNA. RNA Digestion Poly(A) sequences were isolated by nuclease digestion of RNA that had been extracted with phenol/chloroform and washed with lithium chloride. The RNA was digested with pancreatic and T, RNase according to the procedure of Perlman et al. (1973). The nuclease-resistant RNA was extracted with 2 vol of phenol/ chloroform and then with 2 vol of chloroform. The RNA was precipitated with ethanol and electrophoresed on 10% acrylamide gels. Preparation
of Anucleate
Fragments
L. pictus eggs were washed with acidic MF-MBL to remove membranous material, and then layered over a linear gradient constructed from 1.0 M sucrose and a mixture of equal parts 1.0 M sucrose and MF-MBL (Wilt, 1973). The gradient was centrifuged in a Beckman SW 25.1 rotor at 8000 g for 8 min, and then the speed was increased to 25,000g for 12 min. This procedure splits the eggs into nucleate and anucleate fragments, which band at different positions in the gradient. The anucleate fragments were collected by dripping the gradient through a puncture made in the bottom of the tube, and immediately washed in MF-MBL. The fragments were activated by a twostep process. First, they were incubated for
446
DEVELOPMENTAL BIOLOGY
1 min with lo-” M calcium ionophore A23187 (Eli Lily) and then washed with MF-MBL to remove the ionophore. Next, they were suspended for 20 min in hypertonic MBL (Harvey, 1956) and 1O-2 M ammonium chloride. The activated fragments were washed exhaustively with MF-MBL. RESULTS
Isolation
of RNA
A number of controls were performed to insure that pure poly(A1 RNA was isolated from mitochondria in a quantitative manner. Since harsh conditions were needed to homogenize 21-hr embryos (mesenchyme blastula stage), mitochondria were routinely checked for breakage by assaying the postmitochondrial supernatant for isocitrate dehydrogenase, an enzyme located in the mitochondrial matrix. Less than 6% of the enzyme activity was ever found in the supernatant. To monitor any losses of RNA that occurred during the extraction procedure, “C-ribosomal RNA markers were added to the mitochondrial suspension before RNA extraction in some experiments. At least 97% of the 14CRNA was recovered in every experiment. To be certain that each batch of oligo(dT) cellulose we used could effectively separate poly(A) RNA from non poly(A) containing RNA, we chromatographed a mixture of [“Hlpolyadenylic acid (Miles) and “C-ribosomal RNA. Less than 0.2% of the ribosomal RNA was retained on the column in the presence of high salt buffer and 97.6% of the [“Hlpolyadenylic acid was eluted with low salt buffer. Several workers have shown that there is extensive end labeling of tRNA in sea urchin embryos labeled with [“Hluridine (Gross et al., 1965). Therefore, the isolated RNA was always washed with 2 M lithium chloride to remove low molecular weight RNA. Small RNA molecules (e.g., tRNA) and DNA fragments are soluble in this
VOLUME 50. 1976
solution but high molecular weight RNA is not. When the washed RNA was incubated in 0.3 N NaOH for 18 hr at 37”C, it was rendered 98% soluble in cold TCA. Identification RNA
of Mitochondrial
Poly(A)
To determine if sea urchin mitochondria contain poly(A1 RNA we incubated L. pictus embryos with [“Hluridine for 3 hr and extracted the mitochondrial RNA. It was then chromatographed on an oligo(dT) cellulose column to separate any poly(A) RNA from non poly(A) RNA. To prevent any contamination of the poly(A) RNA by non poly(A1 RNA due to adventitious basepairing between them, the RNA samples were always heat-denatured at 65°C for 5 min before being applied to the column. The bound RNA which was subsequently eluted with low salt buffer was apparently free of non poly(A) RNA since when it was passed through the column a second time, more than 99% of it was retained on the column in the presence of high salt buffer. The poly(A1 RNA and non poly(A) RNA were then analyzed by electrophoresis on 2.5% acrylamide gels (Fig. 1). The poly(A) RNA separated into eight distinct species, while the non poly(A) RNA consisted of two species. These latter two probably correspond to the mitochondrial ribosomal RNAs, since electrophoretic profiles of bulk mitochondrial RNA also exhibit bands coincident with these two species. In many cases the determination of RNA molecular weights on aqueous gels is inaccurate. Therefore, the mitochondrial RNA was electrophoresed on 4% formamide gels (Fig. 2). In such gels the secondary structure of RNA is destroyed and electrophoretic mobility is independent of base composition (Pinder et al., 1972). The poly(A) RNA was resolved into eight species on these gels also. Table 1 lists the apparent molecular weights of these eight RNAs. In order to measure the lengths of mitochondrial poly(A1 sequences, mitochon-
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447
165 & 601
40(
20
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40 SIICC no
60
80
FIG. 1. Electrophoretic analysis of mitochondrial RNA. L. picks eggs were fertilized and incubated for 3 hr with 20 @G/ml [:‘Hluridine. Mitochondrial RNA was extracted and chromatographed on oligo(dT) cellulose. The RNA fractions were electrophoresed on 2.5% acrylamide gels, sliced, and counted. (A) Poly(A) RNA; (B) non poly(A) RNA.
drial RNA was extracted from embryos which had been labeled with CZsH]adenosine for 3 hr. After nuclease digestion of the extracted RNA, the resistant fragments were electrophoresed on 10% acrylamide gels. The fragments migrated as a broad peak having a modal value of 4 S (Fig. 3). This represents about 60-70 nucleotides and is in agreement with similar determinations using mitochondria from other sources (Perlman et al., 1973; Avadhani et al., 1973a). Using similar procedures we find that poly(A) sequences of cytoplasmic RNA of sea urchins migrate as a 7 S peak. Isolation
of Mitochondrial
Polysomes
Mitochondrial polysomes were prepared from embryos labeled with [:‘H]leucine in the presence of emetine, a specific inhibitor of nonmitochondrial protein synthesis. The polysomal profile (Fig. 4) exhibits a broad distribution of rapidly sedimenting labeled material, with a large peak at 55 S
and smaller ones at about 110 and 160 S. These probably represent the mitochondrial monomer, dimer, and trimer regions of the gradient. When chloramphenicol, a specific inhibitor of mitochondrial protein synthesis, was added 10 min before the [“Hlleucine pulse there were essentially no counts in the rapidly sedimenting material. Therefore, the labeled material in these structures is the result of actively translating mitochondrial polysomes, and is not due to cytoplasmic protein synthesis or to nonspecific binding of [“Hlleucine. To further demonstrate that the rapidly sedimenting labeled structures are indeed mitochondrial polysomes, we incubated embryos with 150 pg/ml of puromycin 5 min before homogenization. Puromycin will release nascent chains from the polysomes. The data in Fig. 4 indicate that 91% of the labeled material in the structures sedimenting faster than 55 S is absent, indicating that these structures are in fact mitochondrial polysomes.
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DEVELOPMENTAL BIOLOGY
VOLUME 50, 1976 TABLE MOLECULAR
WEIGHTS PoLYCA)
Component
1 OF MITOCHONDRIAL
RNAs Molecular weight” x 10-S 5.6 5.1 4.6 4.2 4.0 3.6 3.4 3.1
LI From Fig. 2(B).
2ocx 50
70
90
Slice no
110
130
FIG. 2. Electrophoretic analysis of mitochondrial RNA on formamide gels. (A) The RNA sample was dissolved in a solution of formamide buffered with 0.001 M sodium phosphate, pH 7.1, and heated to 65°C before being applied to a l&cm gel. (B) The positions of the eight components on the gel are indicated by arrows on the standard curve. This curve was constructed from the electrophoretic mobilities and molecular weights of the large and small mitochondrial ribosomal RNAs. These molecular weights were taken as 5.4 x lo” and 3.4 x 10” in accordance with published values of other animal mitochondrial ribosomal RNAs (e.g., Ojala and Attardi, 1974; Dawid and Chase, 1972).
c
E ci ; ,I 1coc
c
To determine if any of the eight newly synthesized poly(A) RNA species is present in the polysomes, embryos were incubated with [“Hluridine and the mitochondrial polysomes were prepared. The polysomal profile is shown in Fig. 5; the material sedimenting faster than 55 S was collected, made 1% in SDS and 0.5 M in NaCl, chromatographed on an oligo(dT) cellulose column to isolate poly(A) RNA, and electrophoresed on 2.5% gels. The electrophoretic profile (Fig. 6) shows that all eight poly(A) RNA species seen in the mitochondria are also present in the polysomes.
20
40 SIICC no
60
80
FIG. 3. Poly(A) sequences isolated from mitochondrial and cytoplasmic poly(A) RNA. 5’. parpuratus eggs were fertilized and incubated for 3 hr with 20 $.Xml of [:‘Hladenosine. Mitochondrial and cytoplasmic poly(A) RNA was prepared and digested with nucleases. The nuclease resistant fragments were electrophoresed on 10% acrylamide gels, sliced, and counted. 14C 5 and 4 S markers were added to each sample before electrophoresis. O-O, Mitochondrial poly(A) sequences; O-O, cytoplasmic poly(A) sequences.
Anucleate
Fragments
In order to determine the genetic origin (mitochondrial or nuclear) of the poly(A) RNAs found in the mitochondria, sea ur-
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FIG. 4. Mitochondrial polysomal profile of [:‘Hlleucine-labeled embryos. L. picks 18-hr embryos were incubated with 10m4M emetine for 15 min and then 20 pCi/ml of [:‘Hlleucine was added and the embryos incubated for 30 min. Mitochondrial polysomes were analyzed by sucrose gradient centrifugation. O-0, Control polysomes; O--O, polysomes isolated from embryos to which 150 pg/ml of puromycin was added 5 min prior to embryo homogenization; x-x, polysomes isolated from embryos to which 50 pg/ml of chloramphenicol was added 15 min before the [“Hlleucine.
150
50
FIG. 5. Mitochondrial polysomal profile of [:lH]uridine-labeled embryos. L. pi&us 18-hr embryos were labeled with 20 @.X/ml of [:‘Hluridine for 2 hr. The mitochondrial polysomes were then prepared by sucrose gradient centrifugation.
FIG. 6. Poly(A) RNA from mitochondrial polysomes. The material sedimenting faster than 55 S in Fig. 5 (the area enclosed in the bracket) was collected, chromatographed on oligo(dT) cellulose, and the poly(A) RNA was electrophoresed on 2.5% gels.
450
DEVELOPMENTAL BIOLOGY
chin anucleate fragments were analyzed. Sea urchin eggs can be split into nucleate and anucleate fragments, by centrifugation in a sucrose gradient. The anucleate fragments, which contain many of the egg’s mitochondria, can be activated artificially and will cleave several times. During this time the mitochondria synthesize high molecular weight RNA (Chamberlain, 1970; Selvig et al., 1970). This presents an excellent opportunity to study mitochondrial RNA synthesis in the absence of nuclear RNA synthesis. Extensive microscopic examination of the anucleate fragments revealed no contamination from either whole eggs or nucleate fragments. Activated anucleate fragments of L. pictus eggs were incubated with 13Hluridine for 3 hr, and the mitochondrial poly(A) was extracted and electrophoresed on 2.5% acrylamide gels. The electrophoretic profile (Fig. 7) consists of eight peaks of the same size and overall pattern as mitochondrial poly(A) RNA from whole embryos. This demonstrates conclusively that the eight poly(A) RNA species are all transcribed from mitochondrial DNA, not nuclear DNA. Poly(A)
RNA during
Development
In order to detect any changes in the number or amount of poly(A) RNA synthesized during early sea urchin development, mitochondrial RNA was extracted from S. purpuratus embryos pulsed with VH]uridine at various times after fertilization. The RNA was chromatographed on oligo(dT) cellulose and the electrophoretic profiles of the RNA from the various stages of development were compared in Fig. 8. The electrophoretic mobilities of the eight S. purpuratus poly(A) RNAs are slightly larger than the corresponding poly(A) RNAs from L. picks, though the overall pattern is very similar. The mobilities of the non poly(A) RNAs in the two species are identical. As can be seen in Fig. 8, the profiles of the eight RNA spe-
VOLUME 50, 1976
0
1
20
FIG. 7. Poly(A) L. pictus anucleate incubated for 3 hr Poly(A) RNA was electrophoresed on
40 Si1ce no
60
80
RNA from anucleate fragments. fragments were activated and with 20 pCi/ml of [:‘Hluridine. prepared from mitochondria and 2.5% gels.
ties are identical at each of the times studied. Thus there appears to be no qualitative changes in the pattern of mitochondrial RNA synthesis during early sea urchin development. There is, however, a progressive increase in the amount of material larger than peak 1 synthesized as development proceeds. Since it seemed likely that this material represents cytoplasmic contamination of mitochondrial RNA, several controls were run. First, ethidium bromide (a specific inhibitor of mitochondrial RNA and protein synthesis) was added to the embryos 15 min before the [:%Hluridine pulse. The results, shown in Fig. 8, indicate that while the synthesis of RNA larger than peak 1 is only slightly inhibited, the synthesis of RNA smaller than peak 1 is completely inhibited by this compound. Table 2 shows the sensitivity of mitochondrial and cytoplasmic RNA synthesis to ethidium bromide at 3 and 21 hr after fertilization. It is evident that mito-
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Mitochondrial
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451
Synthesis
i
d
28s J
185 I
o-
o-
'0 -
0
r
a
I
C
2occ-
3000-
J 2000.
lOOO-
1000 -
if 0
, 20
10
GO
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FIG. 8. Mitochondrial RNA from different stages of development. S. purpuratus embryos were pulsed for 3 hr with 20 &i/ml of [“Hluridine at 0, 8, and 18 hr after fertilization. The mitochondrial poly(A) RNA and non poly(A) RNA was prepared and electrophoresed on 2.5% gels. (A) Mitochondrial poly(A) RNA from 3-hr embryos; (B) mitochondrial poly(A) RNA from 11-hr embryos; (C) mitochondrial poly(A) RNA from 21-hr embryos; (D) mitochondrial non poly(A) RNA from 21-hr embryos [the mitochondrial non poly(A) RNA from 3-hr and 11-hr embryos is not shown but is exactly the same as that shown for the 21-hr embryos]. O--O, Control poly(A) RNA; O--O, poly(A) RNA from embryos to which 25 pg/ml of ethidium bromide was added 30 min before the [:‘Hluridine pulse.
452
DEVELOPMENTAL BIOLOGY
185 4.
(
_ 20
40 SllCf
no
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80
FIG. 9. Electrophoretic analysis of cytoplasmic poly(A) RNA. S. purpuratus 18-hr embryos were labeled for 3 hr with 20 &i/ml of [:‘H]uridine. RNA was extracted from the postmitochondrial supernatant, chromatographed on oligo(dT) cellulose, and the poly(A) RNA electrophoresed on 2.5% gels.
chondrial RNA synthesis, particularly mitochondrial poly(A) RNA synthesis, is much more sensitive to the drug than cytoplasmic RNA synthesis. Next, the electrophoretic profile of cytoplasmic poly(A) RNA synthesized 21 hr after fertilization was examined. The pattern, shown in Fig. 9, indicates that 98% of this RNA is larger than peak 1. If only 0.1% of this cytoplasmic poly(A) RNA were to contaminate the mitochondria, it would account for the labeled material larger than peak 1 found in the mitochondrial RNA profile at the mesenchyme blastula stage. Finally, if mitochondria are purified by sucrose gradient centrifugation prior to RNA extraction, the material larger than peak 1 is greatly reduced, though not eliminated. The recovery of RNA species smaller than peak 1, however, is completely unaffected. The data from these
VOLUME 50, 1976
three controls indicate that the material larger than peak 1 in the profiles from mitochondria at the later developmental stages is indeed cytoplasmic contamination. The relative amount of newly synthesized mitochondrial RNA which contains poly(A) sequences was also determined at various times during development (Table 3). There is no significant change in the percentage of mitochondrial poly(A) RNA synthesized in stages up to mesenchyme blastula. This is in contrast to the percentage of cytoplasmic poly(A) RNA synthesized, which changes from 13 to 39% as development proceeds. The relative incorporation of [“Hluridine into mitochondrial and cytoplasmic poly(A) RNA at various times during development was also studied (Table 4). The relative rate of incorporation of [“Hluridine into mitochondrial poly(A) RNA remains essentially constant at all times studied, but the incorporation into cytoplasmic poly(A) RNA increases more than loo-fold during development. The rate of [“Hluridine incorporation into mesenchyme blastula mitochondrial poly(A) RNA is slightly larger than at earlier times, but this higher rate is due to contamination by cytoplasmic RNA. If only the poly(A) RNA smaller than peak 1 is counted, the incorporation at this stage is 41,000 cpm/106 embryos. There is no change in the electrophoretic profile of mitochondrial poly(A) RNA or its apparent rate of synthesis during early sea urchin development. However, there is an increase in the amount of labeled RNA found in the mitochondrial polysomes as development proceeds. Figure 10 shows the mitochondrial polysomal profiles prepared from pulse-labeled embryos at 3, 11, and 21 hr after fertilization. Between 11 and 21 hr of development there is a sixfold increase in the labeled material sedimenting faster than 55 S. If this material is chromatographed on collected and oligo(dT) cellulose to isolate poly(A) RNA
ROBERT
DEVLIN
Mitochondrial
Poly(Ai
TABLE SENSITIVITY
OF MITOCHONDRIAL
RNA
2
AND CYTOPLASMIC
RNA SYNTHESIS
Percentage
TO ETHIDIUM
Total RNA RNA
96 38
Poly(A) 99 36
BROMIDE”
RNA inhibition
3-Hr embryos
Mitochondrial Cytoplasmic
453
Synthesis
21-Hr embryos Non poly(A1 92 47
Total
Poly(A1
Non poly(A1
80 13
68 20
74 16
fl At 0 and 18 hr after fertilization 10” S. purpuratus embryos were split into two groups, one of which was incubated for 30 min with 25 Fg/ml of ethidium bromide. Then all embryos were pulsed with 20 &i/ml [“Hluridine for 3 hr. RNA from mitochondria and postmitochondrial supernatants was extracted and washed with lithium chloride. An aliquot from each group was precipitated with cold 10% TCA, filtered, and counted to determine the radioactivity in total mitochondrial and cytoplasmic RNA. The remainder was chromatographed on oligo(dT) cellulose, and aliquots of the poly(A) RNA and non poly(A) RNA were precipitated with TCA and counted.
TABLE
TABLE
3
CHANGES IN THE PERCENTAGE OF MITOCHONDRIAL AND CYTOPLASMIC RNA CONTAINING PoLY(A) SEQUENCES AT DIFFERENT STAGES OF DEVELOPMENT”
3-Hr embryos (%‘o) Mitochondrial Cytoplasmic
11-Hr embryos (70)
53 13
50 20
Radioactivity of poly(A) (cpm/lO” embryos)
21-Hr embryos (o/o) 49 39
* S. purpuratus embryos were labeled for 3 hr with 20 @/ml of [:‘Hluridine. Mitochondrial and cytoplasmic poly(A1 RNA was extracted, washed with lithium chloride, and chromatographed on oligo(dT) cellulose to determine the percentage of total labeled RNA which contains poly(A1 sequences.
there is a corresponding sixfold increase in the amount of labeled poly(A) RNA found in the polysomes. DISCUSSION
In the present study we have demonstrated for the first time the presence of poly(A) containing RNA in mitochondria of sea urchin embryos. This RNA has a poly(A) sequence of 4 S size compared to a 7 S size poly(A) sequence found on cytoplasmic sea urchin poly(A) RNA. In view of the relationship between poly(A) sequences and messenger RNA, and because of the presence of the mitochondrial poly(A) RNA in the polysomes, this RNA almost certainly represents mitochondrial messenger RNA.
4
RELATIVE INCORPORATION OF [“HIURIDINE INTO MITOCHONDRIAL AND CYTOPLASMIC PoLY(A) RNA AT DIFFERENT STAGES OF DEVELOPMENT”
RNA
3-Hr embryos
11-Hr embryos
Mitochondrial (cpm) Cytoplasmic (cm)
42,000
40,000
58,000
12,000
650,000
1,400,000
u S. purpuratus with 20 pCi/ml of cytoplasmic poly(A) phoresed as in Fig. totaled to determine
embryos were labeled for 3 hr [:‘H]uridine. Mitochondrial and RNA was extracted and electro1. The counts on each gel were the relative incorporation rate.
21-Hr embryos
It is unlikely that any of the eight poly(A) RNA species is a degradation product of a larger species, because the electrophoretic profile is very reproducible and there is no evidence of degradation of “C-ribosomal RNA markers added before RNA extraction. It is also unlikely that any of these eight species results from aggregation of smaller RNAs, since the electrophoretic profile observed on aqueous gels is also displayed on gels run in the presence of formamide, which prevents the aggregation of RNA. The possibility that there is a precursor-product relationship between some of the poly(A) RNA species cannot be ruled out since kinetic studies were not done; but since all eight species
454
DEVELOPMENTAL BIOLOGY
0
5
10
VOLUME 50, 1976
15
20
Fractionno
FIG. 10. Mitochondrial polysomal profiles from different stages of development. L. pictus embryos were incubated for 2 hr with 20 &i/ml of [“Hluridine at 0, 8, and 18 hr after fertilization. Polysomes were prepared from the mitochondria by sucrose gradient centrifugation for 3 hr. x-x, Polysomes from 2-hr polysomes from 20-hr embryos. embryos; O-O, polysomes from lo-hr embryos; O---O,
are present in the polysomes, this possibility is unlikely. Therefore, these poly(A) RNAs appear to represent eight unique mitochondrial messenger RNAs. Genetic Origin
of the Poly(A)
RNA
One of the most controversial aspects of mitochondrial biogenesis concerns the genetic origin of mitochondrial messenger RNA. Several lines of evidence suggest that at least some messenger RNAs may be transcribed from nuclear DNA and imported into the mitochondria. The small size of the mitochondrial genome in animal cells has prompted the suggestion that mitochondria have insufficient genetic information to account for the known mitochondrially made proteins and ribosomal RNA (Dawid, 1972). It has been shown that isolated mitochondria are capable of taking up and translating many kinds of RNAs (Swanson, 1972; Kiselev and Gaitskhoki, 1972; Dimitriadis and Georgatos, 1974). It has also been proposed that all of the poly(A) RNA in mitochondria of Ascites cells is of nuclear origin (Avadhani et al., 1973b).
However, our results show that there is no importation of nuclear RNA into mitochondria during early sea urchin development. If any of the eight newly synthesized poly(A) RNA species were synthesized in the nucleus and imported into the mitochondria, it would not appear in the electrophoretic profiles of poly(A) RNA made in anucleate fragments. Because the electrophoretic profile of 13Hluridine-labeled mitochondrial poly(A) RNA from anucleate fragments is identical to the pattern obtained from whole embryos, we feel this constitutes conclusive proof that newly synthesized nuclear RNA is not imported into sea urchin mitochondria during early sea urchin development. Size of the Poly(A)
RNA
Assuming that the molecular weight of the 4.45pm L. pictus mitochondrial DNA molecule (Piko et al., 1968) is 9.5 million daltons, the sum of the eight poly(A) RNA species, the large and small ribosomal RNAs, and the 15 known tRNAs account for 48% of the mitochondrial genome. If the equivalent of only one strand of mito-
ROBERT DEVLIN
Mitochondrial
chondrial DNA is transcribed into functional information, and there is only one copy of each of the poly(A) RNAs in the genome, the RNAs listed above comprise 96% of the functional sequences present on mitochondrial DNA. This would appear to leave no room for any other messenger RNAs or extensive spacer regions on the mitochondrial DNA. Poly(A)
RNA during
Development
Our results indicate there is no change in the electrophoretic profile of newly synthesized mitochondrial poly(A) RNA or non poly(A) RNA during early sea urchin development. Also, there is no change in the percentage of mitochondrial RNA which contains poly(A) sequences as development progresses. Nor is there any apparent change in the relative rate of incorporation of [3H]uridine into mitochondrial poly(A) RNA during early development, although pool sizes were not measured. These findings suggest that virtually the entire functional mitochondrial genome is transcribed at a constant rate throughout early development. Since newly synthesized poly(A) RNA does not appear to reach the polysomes for several hours after fertilization, it appears that translational control mechanisms may be more important in mitochondrial biogenesis during early development than transcriptional mechanisms. It would also indicate that mitochondrial transcription and translation are not coupled, as has been suggested (Chooi and Laird, 1974). I express sincere thanks to Dr. Ronald F. Swanson for his continued support and encouragement. I am also endebted to Dr. Charles Emerson for his numerous helpful suggestions and advice, and to Dr. Irwin R. Konigsberg for his careful editing of this manuscript. This work was supported in part by a Training Program in Developmental Biology (HD 00430) from the National Institutes of Health to the University of Virginia. REFERENCES AVADHANI, N. G., KUAN, P., VANDER LEN, P., and RUTMAN, R. (1973a). Polyadenylic acid sequences
Poly(AA) RNA
Synthesis
455
in mitochondrial RNA. Biochem. Biophys. Res. Commun. 51, 1090-1096. AVADHANI, N. G., BATTULA, N., and RUTMAN, R. (1973b). Messenger RNA metabolism in mammalian mitochondria. Origin of ethidium bromide resistent polyadenylic acid containing RNA in Ehrlich Ascites mitochondria. Biochemistry 12, 4123-4128. AVADHANI, N. G., LEWIS, F., and RUTMAN, R. (1976). Mitochondrial RNA and protein metabolism. Subcellular Biochem., in press. CAVANAUGH, G. M. (19561. In “Formulae and Methods,” p. 55. Woods Hole, Mass. CHAMBERLAIN, J. P. (19701. RNA synthesis in anucleate egg fragments and normal embryos of the sea urchin Arbacia punctulata. Biochim. Biophys. Acta 212, 1833193. CHAMBERLAIN, J. P., and METZ, C. B. (1972). Mitochondrial RNA synthesis in sea urchin embryos. J. Mol. Biol. 64, 593-607. CHOOI, W. Y., and LAIRD, C. D. (1974). Visualization of transcription and translation in mitochondria of Drosophila melanogaster. J. Cell Biol. 63, 60a. DAWID, I. B. (1972). Mitochondrial RNA in Xenopus laeuis. I. The expression of the mitochondrial genome. J. Mol. Biol. 63, 201-216. DAWID, I. B., and CHASE, J. W. (1972). Mitochondrial RNA in Xenopus laeuis. II. Molecular weights and other physical properties of mitochondrial ribosomal and 4S RNA. J. Mol. Biol. 63, 217-231. DIMITRIADIS, G., and GEORGATSOS,J. (1974). Induction of protein synthesis in mitochondria by exogenous RNA. Synthesis of rabbit globin by isolated mitochondria of Tetrahymena pyriformis. FEBS Lett. 46, 96-102. DUESBERG, P., and VOGT, P. (1973). Gel electrophoresis of avian Leukosis and sarcoma viral RNA in formamide: Comparison with other viral and cellular RNA species. J. Vir. 12, 594-599. GROSS, P. R., KRAEMER, K., and MALKIN, L. I. (1965). Base composition of RNA synthesized during cleavage of the sea urchin embryo. Biochem. Biophys. Res. Commun. 18, 569-575. HARVEY, E. B. (1956). In “The American Arbacia and Other Sea Urchins.” Princeton University Press, Princeton, New Jersey. HIRSCH, M., and PENMAN, S. (1973). Mitochondrial polyadenylic acid-containing RNA: Localization and characterization. J. Mol. Biol. 80, 379-391. HIRSCH, M., and PENMAN, S. (1974). The messengerlike properties of the poly(Al+ RNA in mammalian mitochondria. Cell 3, 335-339. KISELEV, O., GAITSKHOKI, V., and NEIFAKH, F. (1975). On the transfer of nuclear RNA into isolated mitochondria. Further evidence for template properties of nuclear RNA taken up by isolated mitochondria. Mol. Cell. Biochem. 6, 149-153. OJALA, D., and ATTARDI, G. (19741. Identification
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and partial characterization of multiple discrete polyadenylic acid containing RNA components coded for by He La cell mitochondrial DNA. J. Mol. Biol. 88, 205-219. PERLMAN, S., ABELSON, H. T., and PENMAN, S. (1973). Mitochondrial protein synthesis: RNA with the properties of eukaryotic messenger RNA. Proc. Nat. Acad. Sci USA 70, 350-353. PIKO, L., BLAIR, D. G., TYLER, A., and VINOGRAD, J. (1968). Cytoplasmic DNA in the unfertilized sea urchin egg: Physical properties of circular mitochondrial DNA and the occurrence of catenated forms. Proc. Nat. Acad. Sci. USA 59, 838-845.
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PINDER, J. C., STAYNOV, D. Z., and GRATZER, W. B. (19741. Electrophoresis of RNA in formamide. Biochemistry 13, 5373-5377. SELVIG, S. E., GROSS, P. R., and HUNTER, A. L. (19701. Cytoplasmic synthesis of RNA in the sea urchin embryo. Develop. Biol. 22, 343-365. SWANSON, R. F. (1971). Incorporation ofhigh molecular weight polynucleotides by isolated mitochondria. Nature (London) 231, 31-34. WILT, F. H. (1973). Polyadenylation of maternal RNA of sea urchin eggs after fertilization. Proc. Nat. Acad. Sci. USA 70, 2345-2349.
