Non-mendelian inheritance of mitochondrial DNA and ribosomal DNA in the myxomycete, Didymium iridis

Mol Gen Genet (1988) 213:37~378

© Springer-Verlag 1988

Non-mendelian inheritance of mitochondrial DNA and ribosomal DNA in the myxomycete, Didymium iridis Margaret E. Sillikcr*, and O'Neil Ray Collins Department of Botany, University of California, Berkeley, CA 94720, USA

Summary. The inheritance of both the mitochondrial DNA (mtDNA) and the nuclear-encoded extrachromosomal ribosomal DNA (rDNA) has been studied in the myxomycete, Didymium iridis, by D N A - D N A hybridization of labeled probes to total DNA at various stages of the life cycle. Both the mtDNA and rDNA populations rapidly become homogeneous in individuals, but there is a qualitative differ- ence in the patterns of inheritance of these two molecules. One parental rDNA type was preferentially inherited in all crosses; selective replication of this molecule is tentati- vely proposed as the mechanism of inheritance. In contrast, either parental mtDNA type could be inherited. Since the inherited population of parental mtDNA molecules are not partitioned into cells in this coenocytic organism, no known mechanism of inheritance can explain the rapid and apparently random loss of one parental mtDNA type in individuals.

Key words: Extrachromosomal inheritance Mitochondrial DNA - Ribosomal DNA - Myxomycete - Non-Mendelian inheritance

Introduction

The present study on organelle inheritance in Didymium iridis (Myxomycetes) is the first report on organelle transmission genetics in a coenocytic organism. Organelle inheritance is typically uniparental, due to uniparental transmission to the zygote or, when biparental zygotes occur, the rapid segregation of parental types during vegetative cell division. Thus, heteroplasmic cells (heterozygous for organelle genes) are rare and unstable (for reviews see Gillham 1978; Birky 1978; Sears 1980). In addition to segregative cell division, intracellular mechanisms may also be impor- tant in establishing homoplasmy; several models have been proposed (Van Winkle-Swift 1980; Birky et al. 1982; Birky 1983). These include (1) genome clustering (which effectively decreases the number of segregating units); (2) selective amplification, replication and/or degradation of different parental types; (3) gene conversion; and (4) selection. Pre- vious studies have dealt with organisms that undergo regu-

* Present address: Department of Microbiology and Immunology, B175, University of Colorado, Health Sciences Center, Denver, CO 80262, USA

Offprint requests to: M.E. Silliker

lar cell division, which accelerates the effects of each of these mechanisms. D. iridis provides a system for studying intracellular mechanisms affecting organelle inheritance, unconfounded by the partitioning of the organelle population under study.

Restriction fragment length polymorphisms for mitochondrial DNA (mtDNA) and ribosomal DNA (rDNA) in D. iridis have been described previously (Silliker 1987). In D. iridis, the nuclear genes that code for the ribosomal RNA are extrachromosomal, on molecules of approximately 20 kb (Silliker 1987). The different restriction patterns for the parental mtDNA and rDNA molecules were used to follow the fate of these molecules in crosses. By studying the inheritance of both mtDNA and rDNA, comparisons of the mode of non-Mendelian inheritance of two different molecules, in two different cellular locations, within a single individual could be made. Since the haploid stage of this organism can be grown indefinitely in liquid culture (Silliker et al. 1988) genetically identical crosses can be made repeatedly for study.

D. iridis is isogamous, with gametes of equal size, which fuse completely to form a zygote. A huge multinucleate coenocytic cell, called a plasmodium, develops. Vegetative segregation via cell division is not an available avenue for sorting out of the mtDNA and rDNA types contributed by both parents, since the plasmodial stage does not divide. In fact, if sufficient nutrients are available, individual cells can grow to be larger than 0.3 m across, though only 1 mm thick. At the time of sporulation, the plasmodium frag- ments into segments which give rise to sporangia, where the spores are delimited and meiosis occurs. Spores germi- nate to haploid amoebae which can be propagated indefinitely or mated to form the diploid.

Examples of intracellular mechanisms that lead to homoplasmy in other organisms were used to generate hypotheses about the mode of inheritance of mtDNA and rDNA in D. iridis. Three general hypotheses were proposed: (1) Early events, prior to mating, or in the zygote, could result in homozygous plasmodia. (2) Individual plasmodia could become homozygous gradually. (3) Plasmodia could remain mixed for mtDNA and rDNA types until events later in the life cycle, i.e. meiosis and sporulation, segregate different parental types. A priori, there was no reason to assume that the inheritance of mtDNA and rDNA would follow the same pattern, or that an identical pattern of inheritance was the result of the same mechanism. Chloroplast DNA (ctDNA) inheritance in Chlamydo-

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monas (Chlorophyta) provides an example of prezygotic and zygotic events that result in the rapid loss of one parental organelle DNA type. The ctDNA associated with one gamete (the rot+ nuclear mating type gene) survives while the ctDNA of the other gamete ( m t - m a t i n g type) is lost after mating (Sager 1972). Though the exact mechanism is still a matter of controversy, there is evidence for degradation of the ctDNA from the m t - parent and selective replication of the ctDNA from the mr+paren t during zygote formation (Gillham 1978; Sager et al. 1984).

The rapid homogenization of mtDNA populations in Saccharomyces cerevisiae has been attributed to random replication and to gene conversion. However, the effective- ness of these mechanisms is partly due to the small mtDNA population size and the partitioning of that population by cell division (Birky et al. 1982). In D. iridis plasmodia these mechanisms could result in a gradual homogenization of the mtDNA and rDNA populations.

There are several examples of selection leading to the loss of one parental mtDNA or rRNA type over time. Loss of one parental mtDNA type has been demonstrated in S. cerevisiae and Paramecium aurelia experimentally by ap- plying strong selection for drug-resistance mutants (Thom- as and Wilke 1968; Linnane et al. 1968; Adoutte and Be- isson 1972). Selection for a particular rDNA type has been demonstrated in Tetrahymena thermophila (Pan et al. 1982; Larson et al. 1986). One rDNA allele type predominates in crosses. Selection is at the level of rDNA replication and minichromosome processing to the mature palindromic form during amplification. The inheritance of rDNA molecules has been studied in Physarum polycephalum, another true slime mold in the same Order as D. iridis. Ferris et al. (1983) have shown that the ratio of parental rDNA types changes in P. polycephalum plasmodia over time, and one particular type consistently predominates. The proportion of progeny inheriting each parental type reflects the proportion of each parental type in the plasmodium just prior to sporulation.

I f mixed mtDNA and rDNA populations persisted in D. iridis plasmodia, sorting out of parental types could occur later, when the cytoplasm is partitioned into spores, or during the vegetative divisions of the haploid amoebae. Sorting out of the different parental mtDNA molecules would be accelerated if like parental genomes were clustered together within mitochondria. There is ultrastructural evidence for mtDNA clustering in D. iridis (Schuster 1965).

To distinguish between the three general modes of inheritance proposed, different stages of the life cycle were analyzed for the presence of the different parental molecules.

Materials and methods

Strains and media. Details of mating and the life cycle of Didymium iridis (Ditmar) Fr. have been reviewed by Collins and Betterley (1982). Compatible heterothallic strains of D. iridis, Hon 1-2 (mating type A 1) and Pan ~ 1 6 (mating type A 7) w e r e used for all crosses. (For further description of these strains see Betterley and Collins 1983.) Preparation of heat-killed bacteria (HKB), pepton~glucose-yeast (PGY) medium and Page's solution is described by Silliker et al. (1988) as well as methods of maintenance of stock cultures and large-scale liquid cultivation of amoebae and microplasmodia.

DAPI staining of amoebae and photography. Haploid amoebae of strains Hon 1 2 and Pan ~ 1 6 were stained with the fluorescent dye, 4,6-Diamidino-2-phenylindole, DAPI, (Sigma D 1388) or 2-(6-Amino-3-imino-3H-xanthen-9-yl) benzoic acid methyl ester, Rhodamine 123 (Sigma R8004, Sigma chemical Company, St. Louis, MO) by mixing 5 gl stationary liquid culture with 5 gl dye solution (15 gg/ml) on a glass slide. A cover slip was added; after 10 min dark incubation the slide was viewed with a Zeiss microscope fitted with IV F1 epifluorescence; excitation illumination was at 365 nm or 546 nm for DAPI and rhodamine, respectively. Photographs were taken with Kodak Ektachrome P800/1600 (process E-6P/push 1) with exposure times of 2 4 s. Cells were randomly chosen for photography and analysis.

Plasmodia for DNA isolation. Crosses were made by com- bining suspensions of amoebae of different mating types on 2% water agar plates (WA) coated with a lawn of heat- killed bacteria (HKB) as described by Silliker et al. (1988). The frequency of mating is low so small plasmodia are widely separated from others and distinguished from unmated amoebae by their larger size and distinct plasmodial morphology with visable shuttle-streaming. Plasmodia could usually be detected within 6 days by using a dissecting microscope (150 x ); they were excised from the agar with a sterile wire and transferred to WA plates (60 x 15 ram) with a streak of HKB (approximately 5- to 8-ram wide). Plasmodia were allowed to migrate away from unmated amoebae, which could potentially mate and fuse with the originally isolated plasmodium. The possibility that small zygotes may have fused prior to isolation cannot be ex- cluded; however, the low mating frequency and early age of isolation make fusion unlikely. At any rate, the small plasmodia are clearly one cell when isolated; the cell that develops is referred to as a zygote clone here. When free of amoebae, they were transferred to (100 x 15 ram) WA plates. Live dilute Escherichia coli suspension was added (0.5 ml; 5 x 10 7 cells/ml) along with approximately 0.25 g sterile rolled oats. When a plasmodium covered a petri dish, it was cut into pie-shaped pieces, which were used to inoculate WA plates sprinkled with sterile oats. Additional transfers were made every 4-6 days. All the cell material was used in subsequent rounds of inoculation to avoid the loss of one parental type of mtDNA or rDNA.

Harvesting plasmodia. Plasmodia were allowed to migrate on the fresh WA away from oats and bacteria, before harvesting. Plates covered with plasmodia were divided in half. The cut edge of each half slab of agar was placed vertically on a tray of fresh agar. The slabs of agar rested against the vertical walls of the tray. This minimized contact between old and new agar surfaces and facilitated the migra- tion of plasmodia on to the clean agar surface. Transfers to WA were made 36 h before harvest to allow most adher- ing bacteria to be ingested. Agar inocula were removed and the ptasmodia were suspended in cold (4 ° C) Page's solution by gently scraping the agar surface with a bent glass rod. Two 40 x 20 cm trays with a 0.5-cm-thick layer of WA agar were used per individual plasmodial clone. Fifty millilitres of Page's solution per tray was sufficient to suspend plasmodia, which were then centrifuged for 3 rain at 500 x g in a Sorvall GLC-1 centrifuge. The super- natant containing bacteria was discarded as was the yellow

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layer of slime on the pellet. The average yield was 1-2 ml of plasmodia, all of which was used for DNA isolation.

Sporangia for DNA isolations. Plasmodia that had migrated away from unmated amoebae (as above) were transferred to another small WA plate with a streak of HKB. When the new streak of HKB was consumed, plasmodia sporu- lated. Entire individual sporangia were used to start new axenic liquid cultures as described by Silliker et al. (1988). These cultures, representing all the viable cells from a sporangium, were used to inoculate 500 ml PGY/HKB liquid medium, maintained on a shaker table (120 rpm; radius of gyration 2.5 cm) at 23 ° C. The resulting amoebae and microplasmodia were used for DNA isolation. The percentage of sporangia sampled from a plasmodium is an estimate of the percentage of the plasmodium sampled since the entire plasmodium is converted into spores at the time of sporulation.

Harvesting sporangiat cultures. Amoebae and microplasmodia from sporangial cultures were concentrated by centrifugation for 7 min at 2000 x g in a Sorvall GSA rotor. The pellets were resuspended in 10-15 ml Page's solution and centrifuged for 3 min at setting 7 of a IEC clinical centrifuge.

Total DNA isolation from plasmodial and sporangial cultures. Total DNA was isolated by a modification of the method of Karrer and Gall (1976). Cells were suspended in 2volumes lysing buffer: 0.5 M EDTA, 0.01 M Tris (pH 9.5) and briefly vortexed. SDS 20% (w/v) was added to a final concentration of 1%. This mixture was heated to 50°C for 20 rain before adding 20 mg/ml self-digested Pronase in 10 mM Tris pH 8.0, I mM EDTA (TE) to a final concentration of 1 mg/ml. Incubation at 50°C was continued for 4 h. The lysate was extracted with one volume of phenol saturated with TE. The aqueous phase was extracted with chloroform and isoamyl alcohol (24: l). CsC1 (1 g per millilitre) was added to the aqueous phase. A refrac- tometer was used to adjust the specific gravity to 1.695 gm/ ml. Bisbenzimide 10 mg/ml (Hoeschst No. 33258, Sigma B- 2883) was added to the CsC1 solution to a final concentration of 100 gg/ml. Samples were centrifuged at 149000 x g in a Beckman SW50.1 rotor for 36 h at 20 ° C. When the gradient was illuminated with long-wave UV-light three fluorescent bands were visible. The upper and lower bands, corresponding to the mtDNA and rDNA bands, respectively, were taken along with substantial contamination from the middle, nuclear, band. The dye was removed by several equal-volume extractions with n-butanol saturated with 5 M NaC1. The DNA solution was dialyzed at 4 ° C against TE, ethanol precipitated and resuspended in TE.

Isolation of purified mtDNA and rDNA for probes. Axeni- cally grown amoebae were concentrated by centrifugation and vortexed with glass beads to break open the cells. Dif- ferential centrifugation was used to separate nuclear and mitochondrial fractions. Nuclear pellets were then treated as described above for whole cell DNA isolation. The rDNA was easily separated as the lowest DNA band after CsC1 equilibrium centrifugation with bisbenzimide as described above. The mitochondrial pellet was lysed with 0.5% Triton X-100. Intact nuclei and mitochondrial membranes were removed by centrifugation before further purl-

Table 1. Number of mitochondria per cell detected by DAPI staining

Strain No. of cells Average no. of Range analyzed mitochondria per cell

Pan 2-16 16 )?= 37.1 + 14.6 18-68

Hon 1-2 19 2= 37.3 _+ 13.9 22-71

fication of the mtDNA on CsCl-bisbenzimide gradients. DNAs were labeled by nick translation (Rigby et al. 1977) with biotin-11-dUTP following the recommendations of the nick translation kit manufacturer (Bethesda Research Lab- oratories, Inc., Gaithersburg, MD).

Restriction endonuclease digestion and electrophoresis. DNA was digested and electrophoresed as described by Taylor and Smolich (1985).

Blotting and DNA/DNA hybridization. Agarose gels were blotted to nylon membranes (Biodyne A 1.2 pm pores, Pall Ultrafine Filtration Corporation, Glen Cove, NY) following the method of Palmer (1986) for bidirectional blots. After blotting, the membrane was baked at 80 ° C for 1 h in a vacuum oven. Prehybridization and hybridization were according to Method I of the membrane manufacturer. Boiled and sheared calf thymus DNA (100 gg/ml) was added to the prehybridization buffer (5 x Deuhardt buffer [Denhardt 1966], 0.9 M NaC1, 50 mM phosphate buffer, pH 7.7, 5 mM EDTA, and 0.2% SDS) and incubated at 65°C for 5 h. Hybridization, using the same buffer, was at 45°C for 12 h. The probe was denatured at 95°C for 10 rain before adding to the hybridization buffer. Post hybridization washes and probe detection were according to the methods described in the BRL DNA Detection Manual (Bethesda Research Laboratories).

Results

Although parallel studies were conducted on both mtDNA and rDNA inheritance in the same individuals for compari- son, the different patterns observed for these molecules are best highlighted by discussing them separately.

Mitochondrial DNA

Number of mitochondria per haploid cell. The mtDNA of amoebae can be stained in vivo with the fluorescent dye, DAPI, and visualized with epi-fluorescence light microsco- py, indicating that each mitochondrion contains many mtDNA copies. Results of counts from two haploid strains used in the study are summarized in Table 1. Averages of the number of mitochondria per cell in each parental strain were identical. The range within each group is large. Since cell division is not synchronized in these cultures, extremes of the range are probably cells just prior to or after mitosis. Counts were made on stationary phase cells, because these cells acted as gametes, i.e., they are competent to mate (Shipley and Holt 1982). There were several possible sources of error in these measurements: (1) Since the DNA in the nucleus is stained as well as the mtDNA the brightly fluorescent nuclei could obscure some mitochondria. This error

Fig. 1. Hybridization of purified Pan 2-16 mtDNA to DNA from individual zygote clones. All lanes were digested with EcoRI and probed with Pan 2-16 mtDNA. Lanes labeled P and H are purified Pan ~16 and Hon 1-2 mtDNA. Lanes labeled z c ( l ~ ) are DNA from three zygote clones. Lanes mc(1, 2) are DNA from many pooled zygotes. Note the mixtures of both parental types in zc2, mcl and mc2

should be evenly distributed between strains. (2) Since amoebae ingest bacteria in vacuoles, bacterial D N A might appear like m t D N A nucleoids. (3) Some mitochondria might be undetected if the number of genomes were too few to be detected by D A P I staining. These errors might be greater in a particular strain.

As an independent check on the D A P I counts, the vital fluorescent dye Rhodamine 123 was also used to stain mitochondria in the parental strains. Unlike DAPI , rhodamine 123 specifically stains mitochondria. The molecular basis of this specificity is unknown but may depend upon the negative electrical potential across the mitochondrial membrane (Johnson et al. 1980). The average numbers of mitochondria per Hon 1-2 cell visualized with either D A P I or Rhodamine 123 stain were compared; the averages 34.6_+ 7.4 and 28.8_+ 9.3, respectively were not statistically different using the Student's t test (alpha = 0.01, N = 10 for each group). Rhodamine 123 staining, though perhaps more accurate, was not used for all of the counts because the red fluorescence was too weak to photograph at expo- sures times short enough to avoid blurring due to move- ment.

Mixtures o f m t D N A were rare in zygote clones. D N A was isolated from 14 zygote clones, 42 60 days after mating. Total m t D N A from strain Pan 2-16 was used to probe blots of plasmodial D N A digested with EcoRI. Mixed parental m t D N A s were detected in only one zygote clone out of the 14 analyzed by D N A - D N A hybridization (Fig. 1, lane zc2). In contrast, D N A isolated from plasmodia derived from many individual zygotes by somatic fusion, were mixed for m t D N A type (Fig. 1, lanes mcl , 2). D. iridis m t D N A inheritance is not analogous to c t D N A inheritance in Chlamydomonas since the m t D N A associated with either

Table 2. Sporangial isolations from zygote clones of Hon 1~

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Pan 2-16 x

Zygote No. of Plasmodia mtDNA rDNA clone sporangia sampled type a type

sampled (%)

PHA 2 14 P P PHB 2 18 H P PHC 3 25 H P PHD 2 22 P P PHE 5 56 H P PHF 6 55 P P PHH 1 6 P P PHI 6 33 P new b

Summary 27 6-56 3 Hon 1-2 0 Hon I-2 5 Pan ~16 7 Pan 2-16

1 new

a Total DNA samples were digested with EcoRI (for mtDNA analysis) or HindIII (for rDNA analysis). EcoRI blots were probed with purified mtDNA from strain Pan 2-16, HindIII blots were probed with purified rDNA, from strain Pan 2-16 b The restriction pattern of this clone, probed with Pan 2-16 resem- bles neither parental rDNA type

mating type could be inherited. Five zygote clones had the Hon 1-2 m t D N A type and eight zygote clones had the Pan 2-16 m t D N A type. Several weeks of growth (approximately 6 weeks) were required before plasmodia were large enough for direct D N A isolation. The data show that by this time, plasmodia were rarely mixed for m t D N A type. In order to understand better the process by which one parental type predominates in individuals, earlier time points were sought.

Loss o f one m t D N A type occurs early in development. To test the hypothesis that mixed m t D N A populations are present in young plasmodia and that one type is gradually lost during development, a strategy was devised to look at the m t D N A population in plasmodia that were younger than could be analyzed by direct D N A isolation from plasmodia. Young plasmodia were forced to sporulate by star- vation. D N A was isolated from clones of cells from individual sporangia. The process of sporulation segregates the m t D N A population into spores and allows components to be cloned apart from mechanisms in the plasmodium which might operate to eliminate different types overtime. Plasmo- dia could be forced to sporulate as early as 20 days after mating. At this time plasmodia were typically 0.5-1.0 cm in diameter and on average produced 12.5 sporangia ( N = 8). Each sporanigal clone was considered a sample from the population of m t D N A molecules in a plasmodium at the time of sporulation. Since the entire plasmodium is converted into sporangia, the percentage of plasmodium sampled is equal to the percentage of sporangia sampled. The sporangial isolations represent data points at least 18 days to I month earlier than could be obtained by isolating D N A directly f rom plasmodia. At this time plasmodia were homozygous for m t D N A type on the basis of blotting and D N A - D N A hybridization methods.

Results from this experiment are summarized in Table 2. Twenty-seven sporangial D N A isolations were made from eight different individually cloned zygotes. One to six spo-

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Fig. 2A, B. Hybridization of purified Pan 2-16 mtDNA and rDNA to total DNA from sporangial cultures PHE (1-5). Purified mtDNA (A) or rDNA (B) from strains Pan 2-16 and Hon 1-2 and total DNA from 5 sporangial cultures from zygote clone PHE were digested with EcoRI (A) or HindIII (B). The gel was blotted and probed with purified Pan 2-16 mtDNA (A) or Pan 2-16 rDNA (B). For each panel: lane 1, purified DNA from Pan ~16; lane 2, purified DNA from Hon 1-2; lanes 3-7, sporangial cultures 1-5 from zygote clone PHE

Fig. 3A, B. Hybridization of purified Pan 2-16 mtDNA and rDNA to total DNA from multiple zygote sporangial cultures. Purified mtDNA (A) or rDNA (B) from strains Pan 2-16 and Hon 1-2 and total DNA from multiple zygote sporangial cultures MZPHI, MZPH2, and MZPH3 were digested with EeoRI (A) or HindIII (B). The gel was blotted and probed with purified Pan 2-16 mtDNA (A) or Pan 2-16 rDNA (B). For each panel; lane 1, purified Pan 2-16 DNA; lane 2, purified Hon 1-2 DNA; lanes 3-5, total DNA from sporangial cultures 1, 2 and 3 from multiple zygote plasmodium MZPHI; lanes 6, 7, total DNA from sporangial cultures 1 and 2 from multiple zygote plasmodium MZPH2, lane 8, total DNA from the sporangial culture from plasmodium from plasmodium MZPH3

rangia were isolated per zygote clone; the estimated range of sample sizes was between 6% and 56%. All sporangia were homozygous for m t D N A type and there was no varia- tion in the m t D N A type between sporangia from one zygote clone. Similar to the results obtained with the plasmodial isolations, either m t D N A type could be maintained, but no mixtures of m t D N A type were found in these clones. A representative blot (Fig. 2A) of total D N A from five different sporangia from zygote clone PHE, probed with Pan 2-16 mtDNA, shows that this zygote clone was homozygous for Hon 1 2 m t D N A type. These data show that the predominace of one m t D N A type is already established 20 days after mating when the plasmodium is no larger than 1.0 cm in diameter.

Mixed mtDNA and rDNA populations are not homogenized by meiosis. An initial hypotheses was that meiosis and sporulation might be the stage at which parental types are sorted out. Results from the plasmodial isolations suggest that loss of one parental type can occur prior to sporulation. This is corroborated by results from the sporangial isola-

tions, given the assumption that the process of meiosis and sporulation does not eliminate one parental type. To test this assumption, developing plasmodia were allowed to fuse repeatedly with other individuals. This is a natural process in D. iridis if plasmodia are isogenic for nuclear vegetative compatibility loci (Clark and Collins 1973; Betterley and Collins 1984). Data have already been presented, which show that plasmodia with mixed m t D N A populations can be obtained by this method. Since plasmodia were allowed to sporulate on the original cross plate, i.e. plasmodia were not separated from mating amoebae, older plasmodia were free to fuse with younger, newly formed plasmodia. Hence a sporangium could result from plasmodia composed of several individuals of different ages.

Three sporangia were analyzed from plasmodium M Z P H I (1-3), two from another plasmodium MZPH2 (1, 2) and one from a small, unisporangiate plasmodium MZPH3. Blots probed with m t D N A are presented in Fig. 3A. Sporangia from plasmodium MZPH1 were mixed for m t D N A type but the relative proportions of parental types form a gradient from the M Z P H I - 1 sporangium, with

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Fig. 4. Hybridization of purified Pan ~16 rDNA to total DNA from six individual zygote clones. All lanes were digested with HindIII and probed with purified Pan 2-16 rDNA. Lanes labeled P and H are Pan 2-16 and Hon 1-2 rDNA, respectively. Lanes 1-6 represent DNA from six individual zygote clones. Contaminants in plasmodial preparations cause the DNA to migrate more slowly

predominantly Hon 1 2 mtDNA, to the M Z P H I - 3 sporangium, where Hon 1-2 mtDNA is barely perceptible. It is interesting that these sporangia were formed in a line of five sporangia along the leading edge of a plasmodium. They represent alternate sporangia. MZPH2-t , 2 were also taken from a linear series of five sporangia from one plasmodium. They represent the first and third sporangia in the series. MZPH2-1 is mixed for mtDNA type but MZPH2-2 is homozygous for the Pan 2-16 mtDNA type. These results show that if mixed populations of mtDNA molecules are present in plasmodia they can be transmitted through the sporangia. No conclusions can be made about whether individual spores were homo- or heterozygous but no one mtDNA type of molecule is systematically eliminated by the process of meiosis and sporulation. These data support the assumption of the previous experiment that cells cloned from sporangia will have mtDNA representative of the plasmodial mtDNA population.

Ribosomal DNA inheritance

One parental rDNA type is preferentially inherited. DNA from 11 plasmodial zygote clones was digested with HindIII, which allows Hon 1-2 and Pan 2-16 rDNA types to be easily distinguished. The Pan 2-16 rDNA type was evident on all ethidium bromide stained gels; Hon 1 5 rDNA was not detected. Six of these zygote clones were further analyzed by blotting and D N A - D N A hybridization but no Hon 1-2 rDNA was detected (Fig. 4). In contrast to the mtDNA, one parental rDNA type is preferentially inherited.

The same 27 sporangial isolations analyzed for mtDNA inheritance were examined for rDNA inheritance by blot-

ring and D N A - D N A hybridization to a Pan 2-16 rDNA probe. The data, summarized in Table 2, show that the Pan 2-16 rDNA type predominated in all sporangial isolations with the exception of sporangial isolate PHI where a novel rDNA molecule was detected. A representative blot of total DNA from five sporangia from plasmodium PHE, digested with HindIII and probed with Pan 2-16 rDNA is shown in Fig. 2B. Minor amounts of Hon 1-2 rDNA are present in lanes 1 and 2; however, Pan ~ 1 6 rDNA predominates. The presence of Hon 1-2 rDNA in PHE 1 and 2 shows, unequivocally, that the Hon 1-2 rDNA is transmitted to zygotes despite the preponderance of Pan 2-16 rDNA. The inheritance of Hon 1 2 mtDNA and Pan 2-16 rDNA, in zygote clone PHE, shows that the inheritance of mtDNA and rDNA is completely independent.

Sporangium MZPH2-1, from a multiple zygote plasmodium, was clearly mixed for rDNA type (Fig 3B). This shows that mixed rDNA populations can be transmitted through sporulation. This result is in agreement with the findings of Ferris et al. (1983); working with P. polycephalum they showed that the proportion of progeny inheriting one parental rDNA type was a reflection of the proportion of that rDNA type in the parent population. In a total of 43 isolations from plasmodia and sporangia the Pan ~ 1 6 rDNA always predominated.

Discussion

The inheritance of two non-chromosomal molecules, the mtDNA and the nuclear-encoded rDNA, was studied in D. iridis. This is the first report of mtDNA inheritance in an organism where, due to its coenocytic structure, the mitochondrial population is not partitioned by cell division during growth. Not surprisingly, the mtDNA and rDNA, which occur in two different cellular locations, are inherited differently. Given that both gametes contribute many copies of both the mtDNA and rDNA to zygotes but plasmodia rapidly become homozygous for both molecules, by what pathway or process is one parental molecular type lost? Differences in the pattern of inheritance of these molecules require that they be discussed separately.

Ribosomal DNA inheritance

Vogt and Braun (1976) estimated that as many as 100-200 copies of rDNA molecules are present per P. polycephalum nucleus. This is probably a reasonable estimate for D. iridis also, based on the relative proportion of rDNA in total DNA isolations. Yet, despite the large number of rDNA molecules contributed to the zygote by the equal size gametes, mixed rDNA populations were rare. Homoge- neity of rDNA molecules was rapidly established and always in favor of the Pan 2-16 rDNA parental type in the cross studied.

The complete predominance of one rDNA type, Pan 2- 16, argues that this molecule has a selective advantage over the Hon 1-2 rDNA type in this genetic background. A test of this hypothesis would be to cross these strains with other strains. Slower homogenization of the rDNA population would be expected in plasmodia where rDNA molecules compete more equally. Working with P. polycephalum, Fer- ris et al. (1983) have described a cross where one particular parental rDNA type progressively decreased during plas-

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modial growth, but they were still able to detect mixed populations of rDNA molecules in 25-week-old plasmodia. Based on a random replication model developed to explain plasmid incompatibility in bacteria (Novick and Hoppen- steadt 1978), Ferris et al. (1983) calculated that 4 months of plasmodial growth would be required for half the nuclei to be fixed for one rDNA type. Clearly, random replication alone cannot explain the loss of one parental rDNA after 2-3 weeks of growth, nor can it explain the consistent maintenance of one of the parental rDNA types. In 43 plasmodial and sporangial isolations the Pan ~ 1 6 rDNA always persisted.

A hierarchy of rDNA inheritance in the macronucleus of T. thermophila has been established for several possible mating combinations (Larson et al. 1986). Sequence differences in the origins of replication of these molecules probably account for the differential replication and therefore unequal maintenance of the these molecules. It would be interesting to see if similar differences could be detected between D. iridis rDNA molecules.

It should be noted that plasmodia apparently homozygous for the Pan 2-16 rDNA type could contain Hon 1-2 rDNA. There is a limited to detection by the blotting and D N A - D N A hybridization technique. (Minor amounts were detected in two of the PHE sporangia.) However, given that the rDNA population is so biased that one parental type is undetectable, or nearly so, it is difficult to see how that minor species would not be lost during random replication and turnover. Ferris et al. (1983) proposed that if mixed populations of rDNA molecules are found in spores of P. polycephalum only one rDNA molecule may be chosen for replication. In this context, minor amounts of an rDNA type might be transmitted effectively to progeny if chosen for amplification. The present work does not provide any evidence concerning individual spores, but it points to the possible significance of the method of inheritance proposed by Ferris et al. (1983). The complete conversion of one plasmodium, PHI, to a novel rDNA molecule, which presumably would be rare initially, suggests that there is some replication bottleneck in the plasmodium.

The presence of both Hon 1-2 and Pan 2-16 rDNA in a sporangium from multiple zygote plasmodium MZPH2-1 indicates that both parental rDNAs get into the zygote even though Hon 1-2 rDNA was rarely detected in plasmodia or sporangia from individually cloned zygotes. The presence of a significant amount of Hon 1-2 rDNA was interpreted as being the result of plasmodial fusions followed by early forced sporulation. Normally, sufficient time for selection of the Pan 2-16 rDNA probably occurs before an individual plasmodium is capable of sporulating. The fusion between different age plasmodia and subsequent sporulation may have "rescued" the Hon 1-2 rDNA type. Once this rDNA was segregated away from the Pan 2-16 rDNA type in spores it would not have to compete, allowing its cloning and detection.

Mitochondrial DNA inheritance

Inheritance of mtDNA in D. iridis cannot be explained by selection since either parental type can survive, just not in the same individual. It is unlikely that gene conversion could be responsible for converting entire genomes (Birky et al. 1982). However, if gene conversion was the mecha-

nism driving homogenization of the mtDNA population a high frequency of intermediate molecular types would be predicted. In D. iridis there was no evidence for gene conversion (or recombination). A restriction-modification system like the one proposed in Chlamydomrnonas (Sager 1972; Sager et al. 1984) would presumably have to be established by a random process in the zygote.

Random replication and turnover might be a reasonable explanation except that the loss of one parental type would occur too slowly to fit the experimental results. Birky (1983) has concluded from computer simulations that the rate of random drift depends on the number of genomes per cell and that this rate is slow for cells with greater than 100 ge- home copies. The rate of drift is accelerated by mechanisms, such as biased replication and degradation, that effectively lower the population size. The number of mtDNA (and rDNA copies) per D. iridis zygote is large. Ultrastructural studies (Schuster 1965) showed that D. iridis mitochondria have a dense core of DNA. It has been estimated that P. polycephalum, which has a similar mitochondrial morphology, has approximately 30-40 molecules per mitochondrion (Kawano et al. 1982). As suggested by Birky (1978), Polya's urn scheme (Feller 1957) could serve as a model for random replication of mtDNA molecules where the population is not partitioned; however, there is no provision for random degradation or molecular turnover which must occur. We have used a model for plasmid incompatibility in bacteria (Ishii et al. 1978) to calculate the rate of random drift. This model assumes random replication of molecules and random assortment to daughter cells. Partitioning in this case, can be thought of as random and fairly drastic degradation; this would then to over-estimate the speed of fixation. The model predicts that 90% segregation will be attained in 120 generations starting with 60 molecules, half contributed from each parent. Given a cell cycle time of 8 h, it would require 40 days for 90% segregation of mtDNA molecules. Actually this grossly underestimates the initial conditions in D. iridis, since evidence presented from DAPI staining showed that each gamete contained approximately 35 mitochondria each with clusters of mtDNA molecules. The data show that plasmodia are homozygous for mtDNA type in 20 days. We conclude that random replication and turnover, alone, cannot account for the rapid loss of one mtDNA type.

All of the intracellular mechanisms proposed for homo- genizing organelle genomes, as presented in the introduction, may operate in concert with random drift to speed the loss of one parental mtDNA type. The DNA core in D. iridis mitochondria is strong evidence for genome clustering. Biased segregation would not occur since mitochondria are not partitioned into different cells, however, the clustering of like genomes might bias replication. Birky (1983) has suggested that the progeny of a newly replicated molecule may be the most likely to be replicated next, due to their proximity to the polymerase. If polymerases are rare, the concentration of one type of molecule in a mitochondrion could bias replication. Kuroiwa et al. (1978) have described a regular mitochondrial division cycle in plasmodia of P. polycephalum : their evidence suggests that mtDNA replication and mitochondrial division is uniform. Other workers report contradictory results (reviewed by Evans 1982); the critical stage to study mtDNA replication, from the standpoint of inheritance, would be in the zygote. At this stage, selective amplification or degradation of one

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molecular type could provide the bias for r andom replica- t ion and turnover to work on.

Several mechanisms may be involved in the observed rapid homogenizat ion of the m t D N A popula t ion , and their roles can be experimental ly dissected. Studies on m t D N A replication, par t icular ly in the zygote will be of interest. Hon 1-2 and Pan 2-16 should be crossed with addi t ional strains to determine general rules of inheri tance in D. iridis.

A n intriguing thought is that nuclear genes may play a role in regulat ing m t D N A inheritance. The dependence of the mi tochondr ia l genome on the nuclear genes is well documented (Borst and Grivell 1978; Fox 1986). In P. aurelia, there is evidence that interspecies mi tochondr ia l compat ibi l i ty is under the control of nuclear genes (Beale and Knowles 1976). Eberhard (1980) proposed that the par- tial uncoupl ing of organelle D N A replicat ion from nuclear D N A may result in in t ra-organismal compet i t ion between both nuclei and organelles and between organelles. Nuclei may regulate organelle inheri tance so that intra-organel le compet i t ion is eliminated. Fur ther genetical analysis will be necessary to determine the impor tance of the nuclear genome in D. iridis m t D N A inheritance.

I f the pat tern of inheri tance repor ted here proves to be the general pa t te rn for D. iridis, the rate of f ixation of m t D N A and r D N A molecules would be more rapid than for nuclear genes (Birky et al. 1983), which would have interesting consequences for popula t ion studies. I f r D N A inheri tance is always biased towards one parent in a cross, the rate of f ixation for r D N A molecules will be faster than for m t D N A molecules, however, recombinat ion between r D N A molecules would have the tendency to slow fixation. A compar ison of relatedness between strains within one popula t ion by analysing restriction fragment length polymorphisms o f both these molecules would be an interesting test of this hypothesis.

Acknowledgements. The authors gratefully acknowledge the assis- tance of Dr. John W. Taylor and Dr. Elizabeth H. Blackburn in reading and commenting on this manuscript. Sincere thanks to Dr. Georgiana May for many helpful discussions. This work was supported by grant @BSR-8415893 from the National Science Foundational to ORC.

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Communicated by R.G. Her rmann

Received July 9, 1987 / December 29, 1987

Non-mendelian inheritance of mitochondrial DNA and ribosomal DNA in the myxomycete, Didymium iridis

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