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Maternal and cytoplasmic inheritance of mitochondrial DNA in Xenopus
DEVELOPMENTAL
29,
BIOLOGY
Maternal
152-161(1972)
and Cytoplasmic IGOR
Department
B.
DAWID
Inheritance in Xenopus AND
ANTONIE
of Mitochondrial W.
of Embryology,
BLACKLER
Carnegie Institution of Washington, Baltimore, Biological Sciences, Cornell Uniuersity Ithuca, New Accepted
April
DNA
Maryland York 14850
21210 and Diuision
of
3, 1972
The inheritance of mitochondrial DNA (mtDNA’) has been studied in the progeny of an interspecific cross of the amphibians Xenopus laeuis and Xenopus mulleri. The mtDNAs of these two species were distinguished by molecular hybridization with cRNA transcribed from the mtDNA of the two species. F, hybrid frogs from either reciprocal cross always contained only maternal mtDNA. Therefore, the mitochondria in somatic cells of the progeny are derived from the numerous mitochondria in the egg. Large oocytes in F, hybrid frogs, or embryos obtained by backcrossing female F, hybrids to parental animals, contained mtDNA derived from the original female parent. These experiments show that mtDNA is inherited cytoplasmically and maternally in Xenopus. Molecular hybridization of nuclear DNA and cRNA transcribed from mtDNA was used to test the question whether copies of the mitochondrial sequences exist in the nucleus. Contamination of nuclear DNA preparations by cytoplasmic mtDNA was excluded by looking for paternal mtDNA sequences in nuclear DNA preparations from hybrid animals. Since no paternal sequences were found we conclude that the nucleus does not contain a “master copy” of the mitochondrial sequences.
the mtDNA pool. Such a hypothesis has been implied by Bell and Miihlethaler (1964), who proposed that during oogenesis in the fern Pteridium aquilinum mitochondria are derived from the nucleus. According to such a hypothesis, mtDNA would replicate within the mitochondria during growth and proliferation of somatic cells, but would be derived from a nuclear copy (or copies) in early gametogenesis. Since there is little doubt that mtDNA replicates autonomously in somatic cells, its inheritance should be uniparental depending on the relative contributions of egg and sperm to the mitochondriai population of the zygote. Eggs of all animals studied accumulate many mitochondria and much mtDNA (Dawid, 1972a) while sperm contain much fewer mitochondria. In Xenopus hevis an egg contains more than 10’ mtDNA molecules (calculated from Dawid, 1966) while each sperm is estimated to contain about 100 molecuIes (Dawid, unpublished). Most or all of the few sperm mitochondria do not proliferate
INTRODUCTION
The genetic autonomy of mitochondria has been demonstrated in fungi by the study of cytoplasmically inherited mutations which reside in mitochondrial DNA (mtDNA’) (Mounolou et al., 1966; Thomas and Wilkie, 1968; Bolotin et al., 1971) and by the maternal inheritance of mtDNA in an interspecific cross in Neurospora (Reich and Luck, 1966). Similar studies have not been reported in animals, but it is known that mtDNA replicates within the mitochondria of different animal cells (Kirschner et al., 1968; Kasamatsu et al., 1971). The question remained open whether animal mtDNA is continually transmitted from one generation to the next within the mitochondria themselves, or whether a “master copy” within the nucleus is replicated at some time during the life cycle to repopulate 1 Abbreviations: mtDNA, mitochondrial DNA; cRNA, complementary RNA synthesized in uitro with Escherichia coli RNA polymerase; mt-cRNA, cRNA transcribed from mtDNA; SSC, 0.15 M sodium chloride, 0.015 M sodium citrate. 152 Copyright All rights
0 1972 by Academic Press. Inc. of reproduction in any form reserved.
DAWID
AND
BLACKLER
Znheritance
in the zygote since they are located within the sperm midpiece which either does not enter the egg at fertilization or is presumed to degenerate later (Ursprung and Schabtach, 1965; Szollosi, 1965). Therefore most of the zygote’s mitochondria must be derived from the egg. The two related species of frogs, X. laevis and X. mulleri, can be crossed to yield viable hybrid progeny (Blackler and Gecking, 1972b). Comparative studies on the nucleotide sequence of the mtDNAs of these two species have shown that their mtDNAs differ sufficiently to be distinguishable by molecular hybridization (Dawid, 1972b). These facts allow us to carry out a genetic study on the inheritance of mtDNA. Hybrid animals derived from X. laevis and X. mulleri have also enabled us to show that nuclear DNA does not contain a copy of the mtDNA sequences. The absence of one complete nuclear copy constitutes a different type of evidence for the genetic autonomy of mtDNA. METHODS
Animals The crossing of X. laevis and X. mulleri to produce hybrid progeny has been described by Blackler and Gecking (197210). In describing crosses we list the female first. Some progeny were bred from “transmission” parents. A “transmission” animal is produced by transplantation of primordial germ cells from a donor to a host embryo at neurulation. It has been shown by morphological and developmental criteria that gametes obtained in this way derive all their characteristics from the donor species (Blackler and Gecking, 1972a, b). Stages of Xenopus embryos are given according to Nieuwkoop and Faber (1956). Cell Fractionation DNA
and
Purification
of
Purification of nuclei from erythrocytes. Blood was collected in SSC and the cells
of
mtDNA
in Xenopus
153
pelleted at about 1000 rpm for 5 min. White cells accumulated at the top of the pellet and were removed. Washing in SSC and removal of white cells was repeated three times. The cells were suspended in 0.25 M sucrose, 0.03 M Tris, pH 7.4, 5 mM MgCl,, and lysed with 0.5% Triton X-100. Nuclei were pelleted at 1500 rpm for 10 min and washed twice in the sucrose solution. The pellet was suspended in 1 M sucrose, 5 mM MgCl*, layered over 2 M sucrose, 5 mM MgC12, and centrifuged for 1 hr at 24,000 rpm in the SW 25.1 rotor of the Beckman centrifuge. The pellet of purified nuclei was used for DNA extraction. Purification of nuclei from liver. Livers were minced, rinsed, homogenized in a loose glass-steel homogenizer in the sucrose-Tris-Mg solution described above, and nuclei were purified by the method described for erythrocytes. Prepamtion of whole cell and nuclear DNA. DNA from nuclei was purified according to Brown and Weber (1968). DNA from whole tadpoles or young frogs, from which the gut was removed, was purified by a slightly modified method (Brown et al., 1972). Preparation of mtDNA. DNA to be used as template for the preparation of cRNA was isolated by a modification of the method of Dawid and Wolstenholme (1967). Mitochondria were isolated from whole ovaries by differential centrifugation and exposed to 20-50 pg/ml DNase in the presence of 5 mM MgCI, for 30 min at room temperature. EDTA was added to 5 mM, the mitochondria pelleted, and banded in a linear 0.9 to 2.1 M sucrose gradient. DNA was extracted by the method of Hirt (1967) and banded in CsCl gradients in the presence of 200 pg/ml ethidium bromide (Radloff et al., 1967). The heavy band, consisting of closed circular molecules, was collected, the dye was removed with Dowex 50, and the DNA purified further by equilibrium centrifugation in a CsCl gradient.
154 Complementary
DEVELOPMENTAL
BIOLOGY
RNA and Hybridization
The conditions described by Reeder and Brown (1970) and Dawid (1972b) were used for the preparation of cRNA. In experiments on nuclear-mitochondrial crosshybridization 3H-labeled ATP, UTP, and CTP were used together at the highest available specific activity, 18-26 mCi/pmole. Only GTP was added in the unlabeled form. The specific activity of the product was about 3 x lo7 cpm/pg. Hybridization in the presence of formamide and ribonuclease treatment of filters were carried out as described by Dawid (197213). Where required, the amount of DNA adsorbed to the filters was measured after hybridization as described by Brown and Weber (1968). RESULTS
a. Inheritance
of mtDNA
DNA of X. laevis may be distinguished from the DNA of X. mulleri because their nucleotide sequences differ considerably. The ratio of the two DNAs may be determined in unknown samples by hybridizing the unknown DNAs with a cRNA mixture containing 3H-cRNA transcribed from the DNA of one species and 32PcRNA from the other. Filters containing pure DNA from each species are also hybridized to the same cRNA mixture. Comparison of the isotope ratios of the cRNA bound to the known and unknown DNAs reveals the relative amounts of X. laevis and X. mulleri DNA present. Since the isotope ratios depend on the input ratios and specific activities of the cRNAs, only results obtained in the same experiment can be compared. This method is independent of the amount of DNA adsorbed to each filter, as long as it is sufficient to bind a significant amount of radioactivity. The same method has been used by Brown and Blackler (1972) to distinguish the bulk nuclear and the ribosomal DNAs from the two Xenopus species.
VOLUME
29, 1972
This hybridization assay has been used to analyze both the nuclear DNAs and the mtDNAs from a series of frogs, including individuals from the progeny of six interspecific crosses. Crosses in either direction were tested. For analysis of the nuclear DNAs a doubly labeled mixture of cRNAs transcribed from both nuclear DNAs was used. The cRNA bound to the DNAs from the hybrid frogs had isotope ratios intermediate between those of the parental species (Table l), confirming the fact that the animals were actually hybrids. For analysis of the mtDNAs, the cRNAs were transcribed from mtDNA of both species. When the doubly labeled mtcRNAs were hybridized to pure mtDNA from each species, the isotope ratio in the cRNA bound differed greatly (Table l).* This difference provides great sensitivity for the detection of mtDNA from one species in the presence of the other. For example, as little as 1% X. mulleri mtDNA added to X. laevis mtDNA caused a noticeable change in the isotope ratios (line 3 in Table 1). Another important aspect of the method is that it does not depend on the use of purified mtDNA for analysis of interspecific crosses. Since cRNA transcribed from highly purified mtDNA does not hybridize with pure nuclear DNA (Section c) one may use whole-cell DNA to test for the nature of mtDNA in an unknown sample. The mtDNA component, which in somatic tissues accounts for between 0.1 and 1%’ of the total DNA, hybridizes unimpeded by the large excess of nuclear DNA (Section c). This is important since it is impractical to purify mtDNA from a small number of hybrid tadpoles. Table 1 shows the results obtained. In every case the isotope ratio of mt-cRNA which bound to the DNA from each hybrid animal was close to the isotope ratio *Note that the absolute ratios depend on the types of cRNA used. In a number of experiments not shown, the label was reversed leading to low ratios for X. laeuis and high ratios for X. mulleri.
DAWID
AND
BLACKLER
Inheritance TABLE
INHERITANCE
Animal
Nuc&mr&oNA
E X. laevis x X. mulleri Y Ad X. mulleri x X. laevis C F H E H’
X. laevis x X. mulleri X mulleri x X. laevis backcross to X. mulleri male
155
in Xenopus
1
OF MITOCHONDRIAL
nuclear DNA 3H : =P
X. laevis X. mulleri
of mtDNA
DNA”
Mitochondrial Source
cRNA
to mtDNA 3H : 32P
of DNA
Parental Ovary mtDNA Ovary mtDNA Artificial mixtures X. 1aevis:X. mulleri 100: 1 2O:l Hybrids (Fl),b female x male 0.51 Whole tadpole DNA’ Whole tadpole DNA 0.44 Whole tadpole DNA 0.47 Whole tadpole DNA Whole tadpole DNA 0.50 0.52 Liver mtDNA Second generation’ Ovary mtDNA 0.51 Whole tadpole DNA 1.0 0.23
bound
9.8 * 0.15 (n = 4) 0.083 * 0.001 (n = 4)
8.4 4.7 11 9.8 0.12 0.12 0.10 0.09 10.5 * 0.5 (n = 5) 0.085
a All filters containing the different nuclear DNAs were allowed to react in the same solution of cRNAs transcribed from nuclear DNA; all mtDNA filters were reacted in a solution of mt-cRNA. In both experiments X. laevis cRNA was labeled with 3H, and X. mulleri cRNA with 32P. The values are means of duplicate determinations, except where a standard deviation and number of determinations is given. Whole-cell DNA was used in amounts between 20 and 50 fig per filter, mtDNA between 0.1 and 1.0 pg. The hybridization mixture (see Methods) contained about 500,000 cpm/ml of each cRNA. All hybridization values used to calculate isotope ratios were at least 10 times blank values. b In all hybrids the female parent is written first. Crosses E, Y, A, and C represent pooled DNA from several individuals; samples F and H are mean values of the results with two individual animals. c All F, tadpoles were past the feeding stage, about 4 wk old. d The female in this cross was a “transmission” animal (see text), i.e., an X. laevis female with X. mulleri eggs. e By second generation we mean either large oocytes of F, hybrids H’). The H’ backcross embryos were at stage 38 to 40 (early swimming nation.
of mt-cRNA bound to the mtDNA of the female parent. Two additional crosses and many samples of the DNAs listed in Table 1 were analyzed in several separate experiments with independent preparations of cRNA; the results always showed that maternal mtDNA was present exclusively. We conclude from these experiments that the mtDNA in the progeny is derived from the mtDNA of the egg. Is the mtDNA in the second generation also derived from the original female parent (the “grandmother”)? In other
(cross E) or backcross embryos tadpoles). See text for further
(cross expla-
words, is the inheritance of mtDNA truly cytoplasmic or only maternal for one generation? To answer this question mature oocytes of F, hybrids and backcrosses were examined. X. laevis x X. mullet-i hybrids frequently form no gonads at all and most of the individuals which form gonads are sterile (Brown and Blackler, 1972). Therefore, only two “second generation” examples are available. The first is an X. laevis x X. mulleri hybrid female which contained an ovary with many large oocytes. These oocytes were at various
156
DEVELOPMENTAL
BIOLOGY
stages of development, including many with extensive yolk deposition. In particular, many oocytes contained large Balbiani bodies which are conglomerations of mitochondria and are thought to be the center of mitochondrial proliferation during oogenesis (Balinsky and Devis, 1963). Since we know that the egg’s mtDNA is the template from which the mtDNA in the offspring is derived, these large oocytes yield a fair sample of “second generation” mtDNA. Table 1 shows that this mtDNA was pure X. laeuis; the mixing experiment in Table 1 allows us to conclude that the ovarian mtDNA in this hybrid frog contained, if any, less than 1% X. mulleri mtDNA. The reciprocal experiment was carried out with whole-cell DNA from backcross embryos obtained from an X. mulleri x X. laevis hybrid female that was mated to a normal X. mulleri male. The mtDNA was that of the maternal grandmother which, in this case, was X. mulleri. These results with reciprocal crosses show that mtDNA in somatic cells and in the oocytes of F, frogs is derived from the female parent. It follows that mtDNA in progeny of F, frogs (if they are viable) will be derived from the F, oocytes, and thus from the original female. From these facts an infinite regress argument allows us to conclude that mtDNA is inherited cytoplasmically in these animals. In a variation on the above experiments that inheritance of the mitochondrial rRNA sequences was tested. This was done by hybridizing ovarian mtDNA from hybrid E (Table 1) with a mixture of mitochondrial 3H-rRNA from X. mulleri and 32P-rRNA from X. laeuis. The sequence difference between the rRNAs of the two species is small but easily measurable (Dawid, 1972b). This experiment showed that the hybrid animal contained maternal (X. laeuis) sequences of mitochondrial rRNA only.
VOLUME
29, 1972
b. No Mitochondria Enter the Germ Cells after Neurulation Hybrid A in Table 1 was derived from a “transmission” female. In this experiment X. mulleri germ cells had been transplanted into an X. laevis embryo at neurulation; the transmission animal was raised and mated to a normal X. laevis male. The mtDNA in the progeny was clearly X. mulleri (Table 1). The experiment extends the concept of germ cell autonomy (Blackler and Gecking, 1972a, b) to mtDNA: although the germ cells of the transmission animal developed in an X. laevis host from neurula onward the mtDNA in the egg was derived from the donor. No mtDNA and, by implication, no mitochondria entered the germ cells at any time after neurulation. The same result was obtained with a second transmission animal from a similar cross in an experiment not shown here. c. There Is Less Than One Complete Copy of mtDNA Sequences per Haploid DNA Complement in the Nucleus Genetic autonomy of mtDNA raises the question of whether or not copies of this DNA exist in the nucleus (“master copies”). Previous work showed the absence of generalized homologies between mtDNA and nuclear DNA in X. laevis (Dawid and Wolstenholme, 1968) and the absence of stretches of mtDNA sequences interspersed in the nuclear DNA of yeast (Cohen et al., 1970). These experiments left open the question whether one or a small number of mtDNA sequences exist in the nucleus. Experiments designed to detect low levels of mtDNA sequences took advantage of the finding that the amount of radioactive RNA bound to homologous DNA is a linear function of the DNA input over a certain range (Brown and Weber, 1968; Chase and Dawid, 1972). Nuclear DNA preparations were hybrid-
DAWID
AND
Inheritance
BLACKLER
ized with highly radioactive cRNA transcribed from purified mtDNA and the amount of radioactive cRNA which bound was compared with the amount of cRNA bound by known amounts of mtDNA. Such a standard curve is shown in Fig. 1. The sensitivity of the assay is critical: one copy of mtDNA per haploid genome of X. laeuis (3.3 pg DNA: Dawid, 1965) amounts to 5 x 10m6 of the nuclear DNA. Since about 100 pg of DNA can be adsorbed to a single filter, we must be able to detect less than 0.5 ng of mtDNA. Figure 1 shows that this low level can be detected and that the presence of a large excess of nuclear DNA does not interfere with the hybridization of cRNA with mtDNA. Different nuclear DNA preparations were hybridized with mt-cRNA; in all
I
mtDNA
per
2
filter,
ng
FIG. 1. Linear relation between cRNA hybridization and the amount of mtDNA adsorbed to filters. Different amounts of Xenopus laeuis mtDNA, either alone (filled circles) or mixed with 115 fig of a nuclear DNA preparation from X. mulleri x X laeuis hybrid animals (open circles) were adsorbed to filters and hybridized together in the same solution of cRNA, which is described in Table 3. Hybridization to mtDNA filters was corrected for binding to filters containing 1 pg of E. coli DNA (45 cpm); hybridization to filters containing both mtDNA and nuclear DNA was corrected for binding to filters with 115 pg nuclear DNA only (890 cpm).
of
mtDNA
in Xenopus
157
cases a significant level of hybridization was observed. We were able to show that this hybridization is due to contamination of the nuclear preparations with cytoplasmic mtDNA by making use of the sequence differences between X. laevis and X. mulleri mtDNA and the maternal inheritance of mtDNA. Table 2 shows that the isotope ratios in the cRNA bound to nuclear DNA preparations from the two species are closely similar to the isotope ratios in pure mtDNA. The isotope ratio of the mt-cRNA which bound to DNA from nuclear preparations of hybrid animals was the same as that which bound to nuclear DNA of the maternal species (Table 2). This was true with hybrid frogs derived from either reciprocal cross. If the binding of mt-cRNA to nuclear DNA preparations from hybrid animals was due to true nuclear sequences then both X. laevis and X. mulleri sequences should be present, and the isotope ratios should be intermediate. The observed maternal inheritance shows that the binding was due principally to the presence of cytoplasmic mtDNA which contaminated the nuclear DNA preparations. This is indicated in Table 2 by listing the species of mtDNA found. Table 2 also shows the number of mtDNA copies per haploid genome in the various DNA preparations as determined from calibration curves like the one shown in Fig. 1 and similar curves derived for each experiment. Since the isotope ratios in the nuclear preparations were not identical to the ratios in pure mtDNA, we tested whether a single copy of paternal mtDNA might still be “hidden” in the nuclear DNA from hybrid animals. The last line of Table 2 shows that the addition of one equivalent of paternal mtDNA to the nuclear DNA led to a change in the isotope ratio which was larger than the difference between pure mtDNA and nuclear DNA prepara-
158
HYBRIDIZATION
DEVELOPMENTAL
OF mt-cRNA
WITH
NUCLEAR
DNA
BIOLOGY
DNA
VOLUME
TABLE 2 PREPARATIONS:
mt-cRNA bound 3H : 32P
29, 1972
CONTAMINATION
BY MATERNAL
mtDNA
present in nuclear preparation
Species
Experiment A X. laeois mtDNA X. mulleri mtDNA X. loeois nuclear DNA X. mulleri nuclear DNA Fl (female X. laeuis x male X. mullen) nuclear DNA Experiment B X. kreois mtDNA X. mulleri mtDNA X. laeuis nuclear DNA X. mulleri nuclear DNA Hybrid (female X. mulleri x male X. loeuis) nuclear DNA Hybrid, plus one equivalent of X. lueuis mtDNA
-
17.9 0.13 9.6 0.23 9.9
X. laevis X. mullet-i X. laeuis
26 0.127 16 0.18 0.18 0.29
X. laeuis X. mulleri X. mulleri -
mtDNA”
Copies per haploid genome
9 15 14
5.3 20 16 -
a Experiments A and B tested different DNA preparations. Each experiment was carried out with one mixture of WcRNA transcribed from X. laeuis mtDNA and 3*P-cRNA transcribed from X. mulleri mtDNA. Filters containing different amounts of mtDNA were included to provide standard curves of the type shown in Fig. 1. Nuclear DNA was used in amounts of 60-120 pg per filter, and each type of DNA was run at least in duplicate. See Table 3 for experimental details on Experiment B.
tion. This fact suggests that no paternal copy is present. A more quantitative consideration of this experiment leads to the same conclusion (Table 3). The maternal (X. mulleri) mtDNA which contaminates the nuclear DNA preparations from the hybrid animals binds primarily 32PcRNA. However, even pure X. mulleri mtDNA binds some 3H-cRNA, in this experiment 0.127 times the amount of 32P-cRNA (Table 2). Therefore, the 3HcRNA which is attributable to X. mulleri mtDNA (0.127 x 32P-radioactivity) was subtracted from the 3H-radioactivity bound to hybrid animal nuclear DNA. The residual (corrected) 3H-radioactivity could be representative of X. laevis mtDNA sequences; this 3H-radioactivity was therefore expressed as copies of mtDNA per haploid genome using the calibration curve of Fig. 1. The result (Table
3) is only 0.18 mtDNA copy per haploid genome in the hybrid animals. Even this low value is probably an artifact and not a reflection of the presence of X. laevis mtDNA sequences, since the same calculation for pure X. mulleri nuclear DNA also left a small residue of corrected 3Hradioactivity, corresponding to 0.22 X. laevis mtDNA copies per genome. This residual radioactivity is likely to be the result of an uncertainty regarding the blank values in the hybridization experiments. Considering the large amount of nuclear DNA on the filters it appears that filters containing similar amounts of E. coli DNA are not entirely appropriate blanks. This is the most probable interpretation, since it is unlikely that X. mulleri cells contain X. laevis mtDNA sequences in their nuclei. In any case, the interpretation of the
DAWID
AND
Inheritance
BLACKLER
residual corrected level of 3H-cRNA binding in the experiment of Table 3 is not very important since it amounts to much less than a single copy of mtDNA per haploid genome. It does not affect the conclusion that in the frog the nucleus does not contain a “master copy” of the mitochondrial sequences.
of
Comments on Molecular Hybridization Studies with Nuclear and Mitochondrial DNA Our results point out how difficult it is to avoid cross-contamination of cell fractions if the test is carried out at high sensitivity. According to “general experience” the nuclear fractions used should have been entirely free of mitochondria. Nevertheless, the hybridization test
The Mode of Inheritance of mtDNA The genetic experiments reported in this paper show that mtDNA is inherited
TABLE OF mt-cRNA
Nuclear
DNA
WITH
preparation
Animal
X. laevis X. mulleri Hybrid (female X. male X. laevis)
NUCLEAR
mulleri
x
DNA
159
in Xenopus
showed that these preparations contained between 5 and 20 copies of mtDNA per haploid equivalent, which were maternally inherited and thus clearly were due to cross-contamination. We feel that crosshybridization studies of this type are generally invalid unless they either give a negative result or their validity can be checked in an independent way. Such a check was possible by testing for paternal mtDNA sequences in the interspecific hybrid between X. laevis and X. mulleri, leading to the conclusion that the nucleus of these animals does not contain a single complete copy of mitochondrial sequences.
DISCUSSION
HYBRIDIZATION
mtDNA
3
PREPARATIONS:
mt-cRNA
ABSENCE
bound
OF PATERNAL
(cpm)”
fig
3H
32P
3H corrected”
71 68 115
3366 408 648
211 2270 3648
(3366) 120 185
mtDNA
SEQUENCES
X. laeuis mtDNA’ copies per haploid genome
5.3 (3) 0.22 i 0.06 (5) 0.18 + 0.04 (5)
“X. laeuis ‘H-mt-cRNA (18 x 10” cpm) and X. mulleri ““P-mt-cRNA (12 x lo6 cpm) were mixed in a volume of 8 ml. Filters containing the nuclear DNAs listed, mtDNA containing filters used to derive the standard curve of Fig. 1, and E. coli DNA filters, were hybridized together in this cRNA solution. The values were corrected for binding to filters containing about 70 rg E. coli DNA: 240 cpm for 3H and 112 cpm for azP. Some results from this reaction are shown as Experiment B in Table 2. b These values were corrected for the binding of 3H-cRNA to the X. mulleri mtDNA which contaminates the nuclear preparations from X. mulleri and from hybrid frogs. This was done by subtracting from the 3Hradioactivity the ‘*P-radioactivity times 0.127, because pure X. mulleri mtDNA bound 0.127 times as much 3H as 32P in this experiment (see Table 2 and text). ‘Corrected values of ‘H-cRNA bound were converted to nanograms of X. laeuis mtDNA using the standard curve of Fig. 1. These values were then expressed as copies of mtDNA per haploid genome, i.e., 11.7 x 10” daltons of mtDNA per 3.3 pg of total nuclear DNA. The mean, standard deviation, and number of determinations are listed. If the corrected values of ‘H-cRNA bound are taken as a reflection of X. laeuis mtDNA sequences in the nucleus then only the X. laevis component of the nuclear DNA of hybrid animals should contain these sequences. In this case one should express the number of mtDNA copies with respect to the X. laevis component of the nuclear genome only, i.e., half the total DNA. This calculation yields twice the number of mtDNA copies listed in the table, i.e., 0.36 copy per genome. The value of 0.18 is listed to allow comparison with the value calculated for pure X. mulleri nuclear DNA, where expression per X. laeuis component is impossible.
160
DEVELOPMENTAL
BIOLOGY
maternally and cytoplasmically in Xenopus. The same conclusion has been derived from work in Neurospora (Reich and Luck, 1966) and with respect to cyt.oplasmic inheritance in yeast (Mounolou et al., 1966; Thomas and Wilkie, 1968; Bolotin et al., 1971). These examples from such diverse groups as fungi and vertebrate animals make it probable that cytoplasmic and (where appropriate) maternal inheritance of mtDNA is a universal phenomenon. Mitochondria depend for their biogenesis on the nucleus and cell sap, since most mitochondrial proteins are “imported” (for reviews see Ashwell and Work, 1970; Borst, 1972). Nevertheless these organelles maintain a small amount of genetic information which is restricted to the mitochondria and is not carried through the nucleus during its transmission from cell to cell or generation to generation. The evolution of the sequences in mtDNA, which is relatively rapid and differential in the Xenopus species studied (Dawid, 1972b), therefore occurs in the mitochondria themselves. The phenomenon of maternal inheritance of mtDNA in Xenopus can now be described in some detail. The X. laevis oocyte accumulates a large number of mitochondria (Dawid, 1966); proliferation of these particles appears to be initiated in young oocytes or in oogonia in the Balbiani body (Balinsky and Devis, 1963; AlMukhta and Webb, 1971). We conclude from the continual cytoplasmic transmission of mtDNA that the Balbiani body is likely to arise by the proliferation of one or several of the mitochondria present in the oogonia. The experiments involving “transmission” hybrids (Results, Section b) show that mitochondria do not enter the developing oocyte from other parts of the body. The mitochondria present in the mature egg are sufficient to supply the needs of the embryo for about 2 days of development. A net increase in mtDNA and in
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1972
total mitochondrial mass takes place in swimming tadpoles after stage 38 (Chase and Dawid, 1972). Since the determination of germ cells is accomplished at or before neurulation (Blackler and Fischberg, 1961), long before stage 38, it follows that the small fraction of oocyte mitochondria which become included in the primary germ cells are the progenitors of all oocyte mitochondria of the developing frog (if it is a female), and thus of the mitochondria of the next generation. We thank Dr. Donald D. Brown for the gift of materials and for many suggestions, discussion, and criticism throughout the course of this project; the work reported in this and the preceding paper owes much to his help. We also thank Mrs. M. Rehbert for careful technical assistance, Drs. R. H. Reeder, L. P. Gage, and P. Lizardi and Messrs. R. H. Stern and D. L. Leister for their suggestions in the preparation of the manuscripts. REFERENCES AL-MUKHTA, K. A. K., and WEBB, A. C. (1971). An ultrastructural study of primordial germ cells, oogonia and early oocytes in Xenopus laeuis. J. Embryol. Exp. Morphol. 26, 195-217. ASHWELL, M., and WORK, T. S. (1970). The biogenesis of mitochondria. Annu. Rev. Biochem. 39, 251-290. BALINSKY, B. I., and DEVIS, R. J. (1963). Origin and differentiation of cytoplasmic structures in the oocytes of Xenopus laeuis. Acta Embtyol. Morphol. Exp. 6, 55-108. BELL, P. R., and M~HLETHALER, K. (1964). Evidence for the presence of deoxyribonucleic acid in the organelles of the egg cells of Pteridium aquilinum. J. Mol. Biol. 8, 853-862. BLACKLER, A. W., and FISCHBERG, M, (1961). Transfer of primordial germ cells in Xenopus laeuis. J. Embryol. Eap. Morphol. 9,634-641. BLACKLER, A. W., and GECKING, C. A. (1972a). Transmission of sex cells of one species through the body of a second species in the genus Xenopus. I. Intraspecific matings. Deuelop. Biol. 27,376-384. BLACKLER, A. W., and GECKING, C. A. (1972b). Transmission of sex cells of one species through the body of a second species in the genus Xenopus. II. Interspecific matings. Deuelop. Biol. 27, 385-394. BOLOTIN, M., COEN, D., DEUTSCH, J., NETTER, P., PETROCHILO, E., and SLONIMSKI, P. P. (1971). La recombinaison des mitochondries chez Sacchromyces cereuisiae. Bull. Inst. Pasteur 69, 215-239.
DAWID
AND BLACKLER
Inheritance
BORST, P. (1972). Mitochondrial nucleic acids. Annu. Reu. Biochem. 41, in press. BROWN, D. D., and BLACKLER, A. W. (1972). Gene amplification proceeds by a chromosome copy mechanism. J. Mol. Biol. 63, 75-83. BROWN, D. D., and WEBER, C. S. (1968). Gene linkage by RNA-DNA hybridization. I. Unique DNA sequences homologous to 4s RNA, 5S RNA, and ribosomal RNA. J. Mol. Biol. 34, 661-680. BROWN, D. D., WENSINK, P. C., and JORDAN, E. (1972). A comparison of the ribosomal DNAs of Xenopus laeuis and Xenopus mulleri: the evolution of tandem genes. J. Mol. Biot. 63, 57-$3. CHASE, J. W., and DAWID, I. B. (1972). Biogenesis of mitochondria during Xenopus laeuis development. Deuelop. Viol. 27, 504-518. COHEN, L. H., HOLLENBERG, C. P., and BORST, P. (1970). An analysis of a possible base sequence complementarity of mitochondrial and nuclear DNA in yeast. Biochim. Biophys. Acta 224, 610613. DAWID, I. B. (1965). Deoxyribonucleic acid in amphibian eggs. J. Mol. Biol. 12, 581-599. DAWID, I. B. (1966). Evidence for the mitochondrial origin of frog egg cytoplasmic DNA. Proc. Nat. Acad. Sci. U.S. 56, 269-276. DAWID, I. B. (1972a). Cytoplasmic DNA. In “Oogenesis” (J. D. Biggers and A. W. Schuetz, eds.). University Park Press, Baltimore, Maryland, pp. 215226. DAWID, I. B. (1972b). Evolution of mitochondrial DNA sequences in Xenopus. Develop. Biol. 29, 139-151. DAWID, I. B., and WOLSTENHOLME, D. R. (1967). Ultracentrifuge and electron microscope studies on the structure of mitochondrial DNA. J. Mol. Biol. 28,2X-245. DAWID, I. B., and WOLSTENHOLME, D. R. (1968). Renaturation and hybridization studies of mitochondrial DNA. Biophys. J. 8, 65-81. HIRT, B. (1967). Selective extraction of polyoma
of mtDNA
in Xenopus
161
DNA from infected mouse cell cultures. J. Mol. Biol. 26, 365369. KASAMATSU, H., ROBBERSON, D. L., and VINOGRAD, J. (1971). A novel closed-circular mitochondrial DNA with properties of a replicative intermediate. Proc. Nat. Acad. Sci. U.S. 68, 2252-2257. KIRSCHNER, R. H., WOLSTENHOLME, D. R., and GROSS, N. J. (1968). Replicating molecules of circular mitochondrial DNA. Proc. Nat. Acad. Sci. U.S. 60,1466-1472. MOUNOLOU, J., JACOB, H., and SLONIMSKI, P. P. (1966). Mitochondrial DNA from yeast petite mutants: specific changes in buoyant density corresponding to different cytoplasmic mutants. Biochem. Biophys. Res. Commun. 24, 218-224. NIEUWKOOP, P. D., and FABER, J. (1956). “Normal Table of Xenopus laeuis (Daudin).” North-Holland Publ., Amsterdam. RADLOFF, R., BAUER, B., and VINOGRAD, J. (1967). A dye-buoyant-density method for the detection and isolation of closed circular duplex DNA: the closed circular DNA in HeLa cells. Proc. Nat. Acad. Sci. U.S. 57, 1514-1521. REEDER, R. H., and BROWN, D. D. (1970). Transcription of ribosomal RNA genes of an amphibian by the RNA polymerase of a bacterium. J. Mol. Biol. 51, 361377. REICH, E., and LUCK, D. J. L. (1966). Replication and inheritance of mitochondrial DNA. Proc. Nat. Acad. Sci. U.S. 55, 1600-1608. SZOLLOSI, D. G. (1965). The fate of sperm middlepiece mitochondria in the rat egg. J. Exp. Zool. 159,367378. THOMAS, D. Y., and WILKIE, D. (1968). Inhibition of mitochondrial synthesis in yeast by erythromycin: cytoplasmic and nuclear factors controlling resistance. Genet. Res. 11,3341. URSPRUNG, H., and SCHABTACH, E. (1965). Fertilization in tunicates: loss of the paternal mitochondrion prior to sperm entry. J. Exp. 2001. 159,379384.
29,
BIOLOGY
Maternal
152-161(1972)
and Cytoplasmic IGOR
Department
B.
DAWID
Inheritance in Xenopus AND
ANTONIE
of Mitochondrial W.
of Embryology,
BLACKLER
Carnegie Institution of Washington, Baltimore, Biological Sciences, Cornell Uniuersity Ithuca, New Accepted
April
DNA
Maryland York 14850
21210 and Diuision
of
3, 1972
The inheritance of mitochondrial DNA (mtDNA’) has been studied in the progeny of an interspecific cross of the amphibians Xenopus laeuis and Xenopus mulleri. The mtDNAs of these two species were distinguished by molecular hybridization with cRNA transcribed from the mtDNA of the two species. F, hybrid frogs from either reciprocal cross always contained only maternal mtDNA. Therefore, the mitochondria in somatic cells of the progeny are derived from the numerous mitochondria in the egg. Large oocytes in F, hybrid frogs, or embryos obtained by backcrossing female F, hybrids to parental animals, contained mtDNA derived from the original female parent. These experiments show that mtDNA is inherited cytoplasmically and maternally in Xenopus. Molecular hybridization of nuclear DNA and cRNA transcribed from mtDNA was used to test the question whether copies of the mitochondrial sequences exist in the nucleus. Contamination of nuclear DNA preparations by cytoplasmic mtDNA was excluded by looking for paternal mtDNA sequences in nuclear DNA preparations from hybrid animals. Since no paternal sequences were found we conclude that the nucleus does not contain a “master copy” of the mitochondrial sequences.
the mtDNA pool. Such a hypothesis has been implied by Bell and Miihlethaler (1964), who proposed that during oogenesis in the fern Pteridium aquilinum mitochondria are derived from the nucleus. According to such a hypothesis, mtDNA would replicate within the mitochondria during growth and proliferation of somatic cells, but would be derived from a nuclear copy (or copies) in early gametogenesis. Since there is little doubt that mtDNA replicates autonomously in somatic cells, its inheritance should be uniparental depending on the relative contributions of egg and sperm to the mitochondriai population of the zygote. Eggs of all animals studied accumulate many mitochondria and much mtDNA (Dawid, 1972a) while sperm contain much fewer mitochondria. In Xenopus hevis an egg contains more than 10’ mtDNA molecules (calculated from Dawid, 1966) while each sperm is estimated to contain about 100 molecuIes (Dawid, unpublished). Most or all of the few sperm mitochondria do not proliferate
INTRODUCTION
The genetic autonomy of mitochondria has been demonstrated in fungi by the study of cytoplasmically inherited mutations which reside in mitochondrial DNA (mtDNA’) (Mounolou et al., 1966; Thomas and Wilkie, 1968; Bolotin et al., 1971) and by the maternal inheritance of mtDNA in an interspecific cross in Neurospora (Reich and Luck, 1966). Similar studies have not been reported in animals, but it is known that mtDNA replicates within the mitochondria of different animal cells (Kirschner et al., 1968; Kasamatsu et al., 1971). The question remained open whether animal mtDNA is continually transmitted from one generation to the next within the mitochondria themselves, or whether a “master copy” within the nucleus is replicated at some time during the life cycle to repopulate 1 Abbreviations: mtDNA, mitochondrial DNA; cRNA, complementary RNA synthesized in uitro with Escherichia coli RNA polymerase; mt-cRNA, cRNA transcribed from mtDNA; SSC, 0.15 M sodium chloride, 0.015 M sodium citrate. 152 Copyright All rights
0 1972 by Academic Press. Inc. of reproduction in any form reserved.
DAWID
AND
BLACKLER
Znheritance
in the zygote since they are located within the sperm midpiece which either does not enter the egg at fertilization or is presumed to degenerate later (Ursprung and Schabtach, 1965; Szollosi, 1965). Therefore most of the zygote’s mitochondria must be derived from the egg. The two related species of frogs, X. laevis and X. mulleri, can be crossed to yield viable hybrid progeny (Blackler and Gecking, 1972b). Comparative studies on the nucleotide sequence of the mtDNAs of these two species have shown that their mtDNAs differ sufficiently to be distinguishable by molecular hybridization (Dawid, 1972b). These facts allow us to carry out a genetic study on the inheritance of mtDNA. Hybrid animals derived from X. laevis and X. mulleri have also enabled us to show that nuclear DNA does not contain a copy of the mtDNA sequences. The absence of one complete nuclear copy constitutes a different type of evidence for the genetic autonomy of mtDNA. METHODS
Animals The crossing of X. laevis and X. mulleri to produce hybrid progeny has been described by Blackler and Gecking (197210). In describing crosses we list the female first. Some progeny were bred from “transmission” parents. A “transmission” animal is produced by transplantation of primordial germ cells from a donor to a host embryo at neurulation. It has been shown by morphological and developmental criteria that gametes obtained in this way derive all their characteristics from the donor species (Blackler and Gecking, 1972a, b). Stages of Xenopus embryos are given according to Nieuwkoop and Faber (1956). Cell Fractionation DNA
and
Purification
of
Purification of nuclei from erythrocytes. Blood was collected in SSC and the cells
of
mtDNA
in Xenopus
153
pelleted at about 1000 rpm for 5 min. White cells accumulated at the top of the pellet and were removed. Washing in SSC and removal of white cells was repeated three times. The cells were suspended in 0.25 M sucrose, 0.03 M Tris, pH 7.4, 5 mM MgCl,, and lysed with 0.5% Triton X-100. Nuclei were pelleted at 1500 rpm for 10 min and washed twice in the sucrose solution. The pellet was suspended in 1 M sucrose, 5 mM MgCl*, layered over 2 M sucrose, 5 mM MgC12, and centrifuged for 1 hr at 24,000 rpm in the SW 25.1 rotor of the Beckman centrifuge. The pellet of purified nuclei was used for DNA extraction. Purification of nuclei from liver. Livers were minced, rinsed, homogenized in a loose glass-steel homogenizer in the sucrose-Tris-Mg solution described above, and nuclei were purified by the method described for erythrocytes. Prepamtion of whole cell and nuclear DNA. DNA from nuclei was purified according to Brown and Weber (1968). DNA from whole tadpoles or young frogs, from which the gut was removed, was purified by a slightly modified method (Brown et al., 1972). Preparation of mtDNA. DNA to be used as template for the preparation of cRNA was isolated by a modification of the method of Dawid and Wolstenholme (1967). Mitochondria were isolated from whole ovaries by differential centrifugation and exposed to 20-50 pg/ml DNase in the presence of 5 mM MgCI, for 30 min at room temperature. EDTA was added to 5 mM, the mitochondria pelleted, and banded in a linear 0.9 to 2.1 M sucrose gradient. DNA was extracted by the method of Hirt (1967) and banded in CsCl gradients in the presence of 200 pg/ml ethidium bromide (Radloff et al., 1967). The heavy band, consisting of closed circular molecules, was collected, the dye was removed with Dowex 50, and the DNA purified further by equilibrium centrifugation in a CsCl gradient.
154 Complementary
DEVELOPMENTAL
BIOLOGY
RNA and Hybridization
The conditions described by Reeder and Brown (1970) and Dawid (1972b) were used for the preparation of cRNA. In experiments on nuclear-mitochondrial crosshybridization 3H-labeled ATP, UTP, and CTP were used together at the highest available specific activity, 18-26 mCi/pmole. Only GTP was added in the unlabeled form. The specific activity of the product was about 3 x lo7 cpm/pg. Hybridization in the presence of formamide and ribonuclease treatment of filters were carried out as described by Dawid (197213). Where required, the amount of DNA adsorbed to the filters was measured after hybridization as described by Brown and Weber (1968). RESULTS
a. Inheritance
of mtDNA
DNA of X. laevis may be distinguished from the DNA of X. mulleri because their nucleotide sequences differ considerably. The ratio of the two DNAs may be determined in unknown samples by hybridizing the unknown DNAs with a cRNA mixture containing 3H-cRNA transcribed from the DNA of one species and 32PcRNA from the other. Filters containing pure DNA from each species are also hybridized to the same cRNA mixture. Comparison of the isotope ratios of the cRNA bound to the known and unknown DNAs reveals the relative amounts of X. laevis and X. mulleri DNA present. Since the isotope ratios depend on the input ratios and specific activities of the cRNAs, only results obtained in the same experiment can be compared. This method is independent of the amount of DNA adsorbed to each filter, as long as it is sufficient to bind a significant amount of radioactivity. The same method has been used by Brown and Blackler (1972) to distinguish the bulk nuclear and the ribosomal DNAs from the two Xenopus species.
VOLUME
29, 1972
This hybridization assay has been used to analyze both the nuclear DNAs and the mtDNAs from a series of frogs, including individuals from the progeny of six interspecific crosses. Crosses in either direction were tested. For analysis of the nuclear DNAs a doubly labeled mixture of cRNAs transcribed from both nuclear DNAs was used. The cRNA bound to the DNAs from the hybrid frogs had isotope ratios intermediate between those of the parental species (Table l), confirming the fact that the animals were actually hybrids. For analysis of the mtDNAs, the cRNAs were transcribed from mtDNA of both species. When the doubly labeled mtcRNAs were hybridized to pure mtDNA from each species, the isotope ratio in the cRNA bound differed greatly (Table l).* This difference provides great sensitivity for the detection of mtDNA from one species in the presence of the other. For example, as little as 1% X. mulleri mtDNA added to X. laevis mtDNA caused a noticeable change in the isotope ratios (line 3 in Table 1). Another important aspect of the method is that it does not depend on the use of purified mtDNA for analysis of interspecific crosses. Since cRNA transcribed from highly purified mtDNA does not hybridize with pure nuclear DNA (Section c) one may use whole-cell DNA to test for the nature of mtDNA in an unknown sample. The mtDNA component, which in somatic tissues accounts for between 0.1 and 1%’ of the total DNA, hybridizes unimpeded by the large excess of nuclear DNA (Section c). This is important since it is impractical to purify mtDNA from a small number of hybrid tadpoles. Table 1 shows the results obtained. In every case the isotope ratio of mt-cRNA which bound to the DNA from each hybrid animal was close to the isotope ratio *Note that the absolute ratios depend on the types of cRNA used. In a number of experiments not shown, the label was reversed leading to low ratios for X. laeuis and high ratios for X. mulleri.
DAWID
AND
BLACKLER
Inheritance TABLE
INHERITANCE
Animal
Nuc&mr&oNA
E X. laevis x X. mulleri Y Ad X. mulleri x X. laevis C F H E H’
X. laevis x X. mulleri X mulleri x X. laevis backcross to X. mulleri male
155
in Xenopus
1
OF MITOCHONDRIAL
nuclear DNA 3H : =P
X. laevis X. mulleri
of mtDNA
DNA”
Mitochondrial Source
cRNA
to mtDNA 3H : 32P
of DNA
Parental Ovary mtDNA Ovary mtDNA Artificial mixtures X. 1aevis:X. mulleri 100: 1 2O:l Hybrids (Fl),b female x male 0.51 Whole tadpole DNA’ Whole tadpole DNA 0.44 Whole tadpole DNA 0.47 Whole tadpole DNA Whole tadpole DNA 0.50 0.52 Liver mtDNA Second generation’ Ovary mtDNA 0.51 Whole tadpole DNA 1.0 0.23
bound
9.8 * 0.15 (n = 4) 0.083 * 0.001 (n = 4)
8.4 4.7 11 9.8 0.12 0.12 0.10 0.09 10.5 * 0.5 (n = 5) 0.085
a All filters containing the different nuclear DNAs were allowed to react in the same solution of cRNAs transcribed from nuclear DNA; all mtDNA filters were reacted in a solution of mt-cRNA. In both experiments X. laevis cRNA was labeled with 3H, and X. mulleri cRNA with 32P. The values are means of duplicate determinations, except where a standard deviation and number of determinations is given. Whole-cell DNA was used in amounts between 20 and 50 fig per filter, mtDNA between 0.1 and 1.0 pg. The hybridization mixture (see Methods) contained about 500,000 cpm/ml of each cRNA. All hybridization values used to calculate isotope ratios were at least 10 times blank values. b In all hybrids the female parent is written first. Crosses E, Y, A, and C represent pooled DNA from several individuals; samples F and H are mean values of the results with two individual animals. c All F, tadpoles were past the feeding stage, about 4 wk old. d The female in this cross was a “transmission” animal (see text), i.e., an X. laevis female with X. mulleri eggs. e By second generation we mean either large oocytes of F, hybrids H’). The H’ backcross embryos were at stage 38 to 40 (early swimming nation.
of mt-cRNA bound to the mtDNA of the female parent. Two additional crosses and many samples of the DNAs listed in Table 1 were analyzed in several separate experiments with independent preparations of cRNA; the results always showed that maternal mtDNA was present exclusively. We conclude from these experiments that the mtDNA in the progeny is derived from the mtDNA of the egg. Is the mtDNA in the second generation also derived from the original female parent (the “grandmother”)? In other
(cross E) or backcross embryos tadpoles). See text for further
(cross expla-
words, is the inheritance of mtDNA truly cytoplasmic or only maternal for one generation? To answer this question mature oocytes of F, hybrids and backcrosses were examined. X. laevis x X. mullet-i hybrids frequently form no gonads at all and most of the individuals which form gonads are sterile (Brown and Blackler, 1972). Therefore, only two “second generation” examples are available. The first is an X. laevis x X. mulleri hybrid female which contained an ovary with many large oocytes. These oocytes were at various
156
DEVELOPMENTAL
BIOLOGY
stages of development, including many with extensive yolk deposition. In particular, many oocytes contained large Balbiani bodies which are conglomerations of mitochondria and are thought to be the center of mitochondrial proliferation during oogenesis (Balinsky and Devis, 1963). Since we know that the egg’s mtDNA is the template from which the mtDNA in the offspring is derived, these large oocytes yield a fair sample of “second generation” mtDNA. Table 1 shows that this mtDNA was pure X. laeuis; the mixing experiment in Table 1 allows us to conclude that the ovarian mtDNA in this hybrid frog contained, if any, less than 1% X. mulleri mtDNA. The reciprocal experiment was carried out with whole-cell DNA from backcross embryos obtained from an X. mulleri x X. laevis hybrid female that was mated to a normal X. mulleri male. The mtDNA was that of the maternal grandmother which, in this case, was X. mulleri. These results with reciprocal crosses show that mtDNA in somatic cells and in the oocytes of F, frogs is derived from the female parent. It follows that mtDNA in progeny of F, frogs (if they are viable) will be derived from the F, oocytes, and thus from the original female. From these facts an infinite regress argument allows us to conclude that mtDNA is inherited cytoplasmically in these animals. In a variation on the above experiments that inheritance of the mitochondrial rRNA sequences was tested. This was done by hybridizing ovarian mtDNA from hybrid E (Table 1) with a mixture of mitochondrial 3H-rRNA from X. mulleri and 32P-rRNA from X. laeuis. The sequence difference between the rRNAs of the two species is small but easily measurable (Dawid, 1972b). This experiment showed that the hybrid animal contained maternal (X. laeuis) sequences of mitochondrial rRNA only.
VOLUME
29, 1972
b. No Mitochondria Enter the Germ Cells after Neurulation Hybrid A in Table 1 was derived from a “transmission” female. In this experiment X. mulleri germ cells had been transplanted into an X. laevis embryo at neurulation; the transmission animal was raised and mated to a normal X. laevis male. The mtDNA in the progeny was clearly X. mulleri (Table 1). The experiment extends the concept of germ cell autonomy (Blackler and Gecking, 1972a, b) to mtDNA: although the germ cells of the transmission animal developed in an X. laevis host from neurula onward the mtDNA in the egg was derived from the donor. No mtDNA and, by implication, no mitochondria entered the germ cells at any time after neurulation. The same result was obtained with a second transmission animal from a similar cross in an experiment not shown here. c. There Is Less Than One Complete Copy of mtDNA Sequences per Haploid DNA Complement in the Nucleus Genetic autonomy of mtDNA raises the question of whether or not copies of this DNA exist in the nucleus (“master copies”). Previous work showed the absence of generalized homologies between mtDNA and nuclear DNA in X. laevis (Dawid and Wolstenholme, 1968) and the absence of stretches of mtDNA sequences interspersed in the nuclear DNA of yeast (Cohen et al., 1970). These experiments left open the question whether one or a small number of mtDNA sequences exist in the nucleus. Experiments designed to detect low levels of mtDNA sequences took advantage of the finding that the amount of radioactive RNA bound to homologous DNA is a linear function of the DNA input over a certain range (Brown and Weber, 1968; Chase and Dawid, 1972). Nuclear DNA preparations were hybrid-
DAWID
AND
Inheritance
BLACKLER
ized with highly radioactive cRNA transcribed from purified mtDNA and the amount of radioactive cRNA which bound was compared with the amount of cRNA bound by known amounts of mtDNA. Such a standard curve is shown in Fig. 1. The sensitivity of the assay is critical: one copy of mtDNA per haploid genome of X. laeuis (3.3 pg DNA: Dawid, 1965) amounts to 5 x 10m6 of the nuclear DNA. Since about 100 pg of DNA can be adsorbed to a single filter, we must be able to detect less than 0.5 ng of mtDNA. Figure 1 shows that this low level can be detected and that the presence of a large excess of nuclear DNA does not interfere with the hybridization of cRNA with mtDNA. Different nuclear DNA preparations were hybridized with mt-cRNA; in all
I
mtDNA
per
2
filter,
ng
FIG. 1. Linear relation between cRNA hybridization and the amount of mtDNA adsorbed to filters. Different amounts of Xenopus laeuis mtDNA, either alone (filled circles) or mixed with 115 fig of a nuclear DNA preparation from X. mulleri x X laeuis hybrid animals (open circles) were adsorbed to filters and hybridized together in the same solution of cRNA, which is described in Table 3. Hybridization to mtDNA filters was corrected for binding to filters containing 1 pg of E. coli DNA (45 cpm); hybridization to filters containing both mtDNA and nuclear DNA was corrected for binding to filters with 115 pg nuclear DNA only (890 cpm).
of
mtDNA
in Xenopus
157
cases a significant level of hybridization was observed. We were able to show that this hybridization is due to contamination of the nuclear preparations with cytoplasmic mtDNA by making use of the sequence differences between X. laevis and X. mulleri mtDNA and the maternal inheritance of mtDNA. Table 2 shows that the isotope ratios in the cRNA bound to nuclear DNA preparations from the two species are closely similar to the isotope ratios in pure mtDNA. The isotope ratio of the mt-cRNA which bound to DNA from nuclear preparations of hybrid animals was the same as that which bound to nuclear DNA of the maternal species (Table 2). This was true with hybrid frogs derived from either reciprocal cross. If the binding of mt-cRNA to nuclear DNA preparations from hybrid animals was due to true nuclear sequences then both X. laevis and X. mulleri sequences should be present, and the isotope ratios should be intermediate. The observed maternal inheritance shows that the binding was due principally to the presence of cytoplasmic mtDNA which contaminated the nuclear DNA preparations. This is indicated in Table 2 by listing the species of mtDNA found. Table 2 also shows the number of mtDNA copies per haploid genome in the various DNA preparations as determined from calibration curves like the one shown in Fig. 1 and similar curves derived for each experiment. Since the isotope ratios in the nuclear preparations were not identical to the ratios in pure mtDNA, we tested whether a single copy of paternal mtDNA might still be “hidden” in the nuclear DNA from hybrid animals. The last line of Table 2 shows that the addition of one equivalent of paternal mtDNA to the nuclear DNA led to a change in the isotope ratio which was larger than the difference between pure mtDNA and nuclear DNA prepara-
158
HYBRIDIZATION
DEVELOPMENTAL
OF mt-cRNA
WITH
NUCLEAR
DNA
BIOLOGY
DNA
VOLUME
TABLE 2 PREPARATIONS:
mt-cRNA bound 3H : 32P
29, 1972
CONTAMINATION
BY MATERNAL
mtDNA
present in nuclear preparation
Species
Experiment A X. laeois mtDNA X. mulleri mtDNA X. loeois nuclear DNA X. mulleri nuclear DNA Fl (female X. laeuis x male X. mullen) nuclear DNA Experiment B X. kreois mtDNA X. mulleri mtDNA X. laeuis nuclear DNA X. mulleri nuclear DNA Hybrid (female X. mulleri x male X. loeuis) nuclear DNA Hybrid, plus one equivalent of X. lueuis mtDNA
-
17.9 0.13 9.6 0.23 9.9
X. laevis X. mullet-i X. laeuis
26 0.127 16 0.18 0.18 0.29
X. laeuis X. mulleri X. mulleri -
mtDNA”
Copies per haploid genome
9 15 14
5.3 20 16 -
a Experiments A and B tested different DNA preparations. Each experiment was carried out with one mixture of WcRNA transcribed from X. laeuis mtDNA and 3*P-cRNA transcribed from X. mulleri mtDNA. Filters containing different amounts of mtDNA were included to provide standard curves of the type shown in Fig. 1. Nuclear DNA was used in amounts of 60-120 pg per filter, and each type of DNA was run at least in duplicate. See Table 3 for experimental details on Experiment B.
tion. This fact suggests that no paternal copy is present. A more quantitative consideration of this experiment leads to the same conclusion (Table 3). The maternal (X. mulleri) mtDNA which contaminates the nuclear DNA preparations from the hybrid animals binds primarily 32PcRNA. However, even pure X. mulleri mtDNA binds some 3H-cRNA, in this experiment 0.127 times the amount of 32P-cRNA (Table 2). Therefore, the 3HcRNA which is attributable to X. mulleri mtDNA (0.127 x 32P-radioactivity) was subtracted from the 3H-radioactivity bound to hybrid animal nuclear DNA. The residual (corrected) 3H-radioactivity could be representative of X. laevis mtDNA sequences; this 3H-radioactivity was therefore expressed as copies of mtDNA per haploid genome using the calibration curve of Fig. 1. The result (Table
3) is only 0.18 mtDNA copy per haploid genome in the hybrid animals. Even this low value is probably an artifact and not a reflection of the presence of X. laevis mtDNA sequences, since the same calculation for pure X. mulleri nuclear DNA also left a small residue of corrected 3Hradioactivity, corresponding to 0.22 X. laevis mtDNA copies per genome. This residual radioactivity is likely to be the result of an uncertainty regarding the blank values in the hybridization experiments. Considering the large amount of nuclear DNA on the filters it appears that filters containing similar amounts of E. coli DNA are not entirely appropriate blanks. This is the most probable interpretation, since it is unlikely that X. mulleri cells contain X. laevis mtDNA sequences in their nuclei. In any case, the interpretation of the
DAWID
AND
Inheritance
BLACKLER
residual corrected level of 3H-cRNA binding in the experiment of Table 3 is not very important since it amounts to much less than a single copy of mtDNA per haploid genome. It does not affect the conclusion that in the frog the nucleus does not contain a “master copy” of the mitochondrial sequences.
of
Comments on Molecular Hybridization Studies with Nuclear and Mitochondrial DNA Our results point out how difficult it is to avoid cross-contamination of cell fractions if the test is carried out at high sensitivity. According to “general experience” the nuclear fractions used should have been entirely free of mitochondria. Nevertheless, the hybridization test
The Mode of Inheritance of mtDNA The genetic experiments reported in this paper show that mtDNA is inherited
TABLE OF mt-cRNA
Nuclear
DNA
WITH
preparation
Animal
X. laevis X. mulleri Hybrid (female X. male X. laevis)
NUCLEAR
mulleri
x
DNA
159
in Xenopus
showed that these preparations contained between 5 and 20 copies of mtDNA per haploid equivalent, which were maternally inherited and thus clearly were due to cross-contamination. We feel that crosshybridization studies of this type are generally invalid unless they either give a negative result or their validity can be checked in an independent way. Such a check was possible by testing for paternal mtDNA sequences in the interspecific hybrid between X. laevis and X. mulleri, leading to the conclusion that the nucleus of these animals does not contain a single complete copy of mitochondrial sequences.
DISCUSSION
HYBRIDIZATION
mtDNA
3
PREPARATIONS:
mt-cRNA
ABSENCE
bound
OF PATERNAL
(cpm)”
fig
3H
32P
3H corrected”
71 68 115
3366 408 648
211 2270 3648
(3366) 120 185
mtDNA
SEQUENCES
X. laeuis mtDNA’ copies per haploid genome
5.3 (3) 0.22 i 0.06 (5) 0.18 + 0.04 (5)
“X. laeuis ‘H-mt-cRNA (18 x 10” cpm) and X. mulleri ““P-mt-cRNA (12 x lo6 cpm) were mixed in a volume of 8 ml. Filters containing the nuclear DNAs listed, mtDNA containing filters used to derive the standard curve of Fig. 1, and E. coli DNA filters, were hybridized together in this cRNA solution. The values were corrected for binding to filters containing about 70 rg E. coli DNA: 240 cpm for 3H and 112 cpm for azP. Some results from this reaction are shown as Experiment B in Table 2. b These values were corrected for the binding of 3H-cRNA to the X. mulleri mtDNA which contaminates the nuclear preparations from X. mulleri and from hybrid frogs. This was done by subtracting from the 3Hradioactivity the ‘*P-radioactivity times 0.127, because pure X. mulleri mtDNA bound 0.127 times as much 3H as 32P in this experiment (see Table 2 and text). ‘Corrected values of ‘H-cRNA bound were converted to nanograms of X. laeuis mtDNA using the standard curve of Fig. 1. These values were then expressed as copies of mtDNA per haploid genome, i.e., 11.7 x 10” daltons of mtDNA per 3.3 pg of total nuclear DNA. The mean, standard deviation, and number of determinations are listed. If the corrected values of ‘H-cRNA bound are taken as a reflection of X. laeuis mtDNA sequences in the nucleus then only the X. laevis component of the nuclear DNA of hybrid animals should contain these sequences. In this case one should express the number of mtDNA copies with respect to the X. laevis component of the nuclear genome only, i.e., half the total DNA. This calculation yields twice the number of mtDNA copies listed in the table, i.e., 0.36 copy per genome. The value of 0.18 is listed to allow comparison with the value calculated for pure X. mulleri nuclear DNA, where expression per X. laeuis component is impossible.
160
DEVELOPMENTAL
BIOLOGY
maternally and cytoplasmically in Xenopus. The same conclusion has been derived from work in Neurospora (Reich and Luck, 1966) and with respect to cyt.oplasmic inheritance in yeast (Mounolou et al., 1966; Thomas and Wilkie, 1968; Bolotin et al., 1971). These examples from such diverse groups as fungi and vertebrate animals make it probable that cytoplasmic and (where appropriate) maternal inheritance of mtDNA is a universal phenomenon. Mitochondria depend for their biogenesis on the nucleus and cell sap, since most mitochondrial proteins are “imported” (for reviews see Ashwell and Work, 1970; Borst, 1972). Nevertheless these organelles maintain a small amount of genetic information which is restricted to the mitochondria and is not carried through the nucleus during its transmission from cell to cell or generation to generation. The evolution of the sequences in mtDNA, which is relatively rapid and differential in the Xenopus species studied (Dawid, 1972b), therefore occurs in the mitochondria themselves. The phenomenon of maternal inheritance of mtDNA in Xenopus can now be described in some detail. The X. laevis oocyte accumulates a large number of mitochondria (Dawid, 1966); proliferation of these particles appears to be initiated in young oocytes or in oogonia in the Balbiani body (Balinsky and Devis, 1963; AlMukhta and Webb, 1971). We conclude from the continual cytoplasmic transmission of mtDNA that the Balbiani body is likely to arise by the proliferation of one or several of the mitochondria present in the oogonia. The experiments involving “transmission” hybrids (Results, Section b) show that mitochondria do not enter the developing oocyte from other parts of the body. The mitochondria present in the mature egg are sufficient to supply the needs of the embryo for about 2 days of development. A net increase in mtDNA and in
VOLUME
29,
1972
total mitochondrial mass takes place in swimming tadpoles after stage 38 (Chase and Dawid, 1972). Since the determination of germ cells is accomplished at or before neurulation (Blackler and Fischberg, 1961), long before stage 38, it follows that the small fraction of oocyte mitochondria which become included in the primary germ cells are the progenitors of all oocyte mitochondria of the developing frog (if it is a female), and thus of the mitochondria of the next generation. We thank Dr. Donald D. Brown for the gift of materials and for many suggestions, discussion, and criticism throughout the course of this project; the work reported in this and the preceding paper owes much to his help. We also thank Mrs. M. Rehbert for careful technical assistance, Drs. R. H. Reeder, L. P. Gage, and P. Lizardi and Messrs. R. H. Stern and D. L. Leister for their suggestions in the preparation of the manuscripts. REFERENCES AL-MUKHTA, K. A. K., and WEBB, A. C. (1971). An ultrastructural study of primordial germ cells, oogonia and early oocytes in Xenopus laeuis. J. Embryol. Exp. Morphol. 26, 195-217. ASHWELL, M., and WORK, T. S. (1970). The biogenesis of mitochondria. Annu. Rev. Biochem. 39, 251-290. BALINSKY, B. I., and DEVIS, R. J. (1963). Origin and differentiation of cytoplasmic structures in the oocytes of Xenopus laeuis. Acta Embtyol. Morphol. Exp. 6, 55-108. BELL, P. R., and M~HLETHALER, K. (1964). Evidence for the presence of deoxyribonucleic acid in the organelles of the egg cells of Pteridium aquilinum. J. Mol. Biol. 8, 853-862. BLACKLER, A. W., and FISCHBERG, M, (1961). Transfer of primordial germ cells in Xenopus laeuis. J. Embryol. Eap. Morphol. 9,634-641. BLACKLER, A. W., and GECKING, C. A. (1972a). Transmission of sex cells of one species through the body of a second species in the genus Xenopus. I. Intraspecific matings. Deuelop. Biol. 27,376-384. BLACKLER, A. W., and GECKING, C. A. (1972b). Transmission of sex cells of one species through the body of a second species in the genus Xenopus. II. Interspecific matings. Deuelop. Biol. 27, 385-394. BOLOTIN, M., COEN, D., DEUTSCH, J., NETTER, P., PETROCHILO, E., and SLONIMSKI, P. P. (1971). La recombinaison des mitochondries chez Sacchromyces cereuisiae. Bull. Inst. Pasteur 69, 215-239.
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