| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Nova Southeastern University College of Osteopathic Medicine, 3200 South University Drive, Fort Lauderdale, Florida 33328
Margaret E. Silliker 1
Department of Biological Sciences, 2325 North Clifton Avenue, DePaul University, Chicago, Illinois 60614
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
To test whether the timing of transition to mating competency affected mitochondrial transmission patterns in D. iridis. Reciprocal crosses were made by combining mating compatible strains that differed in their competency to mate. The results were compared to crosses where both mating strains were competent at the time of combining and crosses where somatic fusion of plasmodia was allowed. The results show that the mating competency of the parental strains at the time of confronting a compatible mate does not affect mitochondrial transmission patterns, mating efficiency or the likelihood of biparental inheritance. However the timing of plasmodial formation is delayed when precompetent and competent strains are mated compared to when both strains are competent at the time of mixing. We also observed that somatic fusion of plasmodia did not appreciably increase the incidence of biparental inheritance compared to crosses where individual plasmodia were isolated. These results provide additional evidence of the variable nature of mitochondrial inheritance in D. iridis within crosses and between mating trials.
Key words: biparental and uniparental mitochondrial inheritance, mating induction, mitochondrial transmittance, plasmodial fusion
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
, Partridge and Hurst 1998). However exceptions to the uniparental pattern of inheritance are numerous; in many cases, mitochondrial inheritance is biased toward one parent but some contribution from the other parent is not completely excluded. Birky (1995, 2001) has argued that mitochondrial inheritance should be considered a quantitative trait, where the frequency of inheritance of parental types is measured because the discrete categories of uni- or biparental inheritance are misleading.
Different mechanisms for regulating mitochondrial inheritance are thought to have evolved independently (Birky 1995, Gillham 1994). If uniparental inheritance is biologically advantageous, the life cycle of the plasmodial slime molds (Myxogastria, Olive 1975) presents the greatest challenge to achieving this goal. In these organisms the mating cells are isogamous and contribute equally to the initial organelle population (Silliker and Collins 1988, Moriyama and Kawano 2003). After syngamy mitotic and mitochondrial divisions occur but are not coupled to cell division, so the mitochondrial population cannot be sorted by cell partitioning. Genetically identical diploid plasmodial cells can fuse somatically, providing the opportunity for further mitochondrial mixing. In addition, multiple mating types in heterothallic species preclude a system where one of only two possible sexes is the designated mitochondrial donor.
Despite these challenges, the pattern of mitochondrial inheritance in plasmodial slime molds appears to be toward the uniparental end of the inheritance spectrum. Until recently mitochondrial inheritance in Physarum polycephalum was thought to be uniparental (Kawano et al 1987, Kawano and Kuroiwa 1989, Meland et al 1991) with the donating parent determined by a mating type hierarchy. Moriyama and Kawano 2003 have found cases of biparental inheritance in P. polycephalum, where the ratio of parental mitochondrial types varied from 1 : 1 to 1 : 10–4. In Didymium iridis the pattern of mitochondrial inheritance is also toward the uniparental end of the spectrum, with cases of biparental inheritance showing highly skewed ratios of parental types (Silliker and Collins 1988, Silliker et al 2002). Curiously, there does not appear to be a mating type hierarchy that determines the parental donor in D. iridis. Silliker et al (2002) found these patterns in D. iridis when uniparental inheritance was observed: (i) for some crosses one parent was always the donor, in other cases, (ii) one parent was the favored donor but occasionally the other parent was the donor, in still other cases, (iii) either parent was equally likely to be the donor. These three patterns were called dominant, biased, and random, respectively. Biparental inheritance also was found in some of the groups showing biased inheritance.
The complex pattern of mitochondrial inheritance in D. iridis suggested that, in addition to genetic factors, conditions at the time of mating might influence the parental donor (Silliker et al 2002). This is particularly supported by the observation that the favored parental donor switched from one parent to the other in different trials of the same cross.
We considered factors that might influence mitochondrial inheritance at the time of mating. In D. iridis cells must be both compatible and competent to mate. Compatibility in the A1 mating series is determined by one locus with multiple alleles (Collins 1963, Clark et al 1991). Competence to mate is induced when haploid cells reach a certain density (Shipley and Holt 1982). This transition to mating competence is thought to be triggered by a secreted inducer that accumulates at high cell densities; at low densities mating does not occur (Ross et al 1973, Shipley and Holt 1982, Nader et al 1984). Shipley and Holt (1982) concluded that both partners must be competent for mating to occur but once competency is triggered it is not known whether all cells maintain a constant level of competency over time.
We hypothesized that the differences in parental mitochondrial transmission between different trials of the same cross might be due to differences in mating competence of the parents at the time of mating. In previous studies of mitochondrial inheritance in D. iridis the mating competence of the mating partners was not consistently controlled. Although high cell densities were always combined, some cultures were just entering the competency phase while other cultures were stationary, having obtained mating competency days earlier. These differences might explain the variable results, between and even within trials. In this study the mating parents were manipulated culturally to keep one parent at a precompetent state (by continuous culture at low cell densities), while the other parent was induced to mating competence (by growth at high cell densities). Reciprocal crosses were made where the precompetent and competent parents at the time of mating were switched. A difference in mitochondrial transmission between reciprocal crosses would support the hypothesis that the timing of transition to mating competence influences mitochondrial donor/recipient relationships between parents. We predicted that the parent that had reached competence first would have an advantage in transmitting its mitochondria.
|
MATERIAL AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
). Dr Jim Clark kindly provided strains Hon1–7 (mating type A2) and Pan2–44 (A8) from the Collins’ mating type testers collection (Collins and Betterley 1982). Hon and Pan names are after their country of origin, Honduras and Panama, respectively. Strains Hon1–2 (A1), and Pan2–16 (A7) were obtained directly from Collins’ collections. Haploid strains were maintained in liquid axenic culture in peptone-glucose-yeast (PGY) media supplemented with heat-killed bacteria (HKB), and once mated the diploid plasmodia were maintained on half-strength cornmeal agar (CMA/2) with live bacteria as previously described (Silliker et al 1988, Silliker et al 2002). Growth curves.— – Growth curves were established for each strain of D. iridis so that the timing of the transition to sexual competency could be determined. Cells from stock cultures were measured with a hemocytometer and adjusted to a concentration of 1 x 103 amoebae/ mL in 6.0 mL of PGY, 0.15 mL of HKB. During growth the cultures were aerated on a Cel-Gro Tissue Culture Rotator, 70 rpm, at 23 C. Counts were made for seven consecutive days to establish the log and stationary phases of growth. Larger volume cultures of amoebae were grown at 23 C on a platform shaker at 175 rpm (1.0 mL HKB/50.0 mL PGY).
High and low density cultivation and mating.— – Using the established growth curves, strains of D. irdis were cultivated so they could be crossed at specific densities. Strains grown to a density of 1 x 106 amoebae/mL, hereafter referred to as the high density strain in a cross, were at the end of log phase which corresponds to the onset of mating competency. The low density strains were grown to a density of 1 x 104 amoebae/ mL, which is below the density for a strain to be mating competent.
Low density cells were concentrated by centrifugation at 5445 g for 7 min. Equal numbers of high and low density cells were suspended in 1.0 mL of the growth media of the high density cells at a final concentration of 1 x 106 amoebae/mL. Concentrated bacteria (see below), 0.5 mL, were added and the cells were incubated 1 h at room temperature before plating equal volumes onto four CMA/2 agar plates (100 x 15 mm). Concentrated bacteria were prepared by suspending live Escherichia coli grown on a nutrient agar (Difco) slant tube (15 x 150 mm) in 4.0 mL of sterile distilled water. The plates were observed daily with a dissecting microscope (250x) until small plasmodia were visible. The mean plasmodial density per cm2 was calculated by counting the number of plasmodia in six squares of a grid (1 cm2 each) placed under the plate. Individual plasmodia were excised on a block of agar with a sterile needle and transferred to a fresh CMA/2 plate (60 x 15 mm). Within a day the plasmodia migrate away from the agar block and any remaining amoebae. Isolated plasmodia were transferred to a CMA/2 plate (100 x 15 mm) coated with concentrated bacterial suspension. Once the plasmodia covered the plate, or had appeared to consume all the bacteria, it was harvested for DNA isolation (usually 5–7 d after the plasmodia is excised from the cross plate).
Equal density and somatic fusion crosses.— – Amoebal cultures were grown several days to late log phase densities. Equal numbers of cells were combined in a final volume of 1.0 mL to which 0.5 mL concentrated bacteria was added. After 1 h of incubation at room temperature the cells were plated as described above. Individual plasmodia were isolated for the equal density crosses. For the somatic fusion crosses the remaining plasmodia were allowed to fuse on the cross plate.
Amoebal DNA isolation.— – To isolate amoebal DNA a 50 mL PGY/HKB culture (1 x 106 amoeabe/mL) was pelleted for 5 min at 5445 g, 4 C. Lysis buffer, 5.0 mL, (10 mM Tris and 0.1 M EDTA, pH 8.0, 0.5% SDS) was added to the pellet and gently pipetted to disperse it. Proteinase K, 25 µl (20 mg/mL), was added and the mixture was incubated at 50 C for 30 min to 1 h. The lysate was extracted with an equal volume of chloroform : isoamyl alcohol (24 : 1). DNA was precipitated from the aqueous phase by addition of 2/3 volume isopropanol. The DNA pellet was suspended in 400 µL of TE (10 mM Tris, 1 mM EDTA, pH 8.0) and RNAse A (final concentration 12.5 µgm/mL) and incubated at 37 C for 30 min. After precipitation the DNA was suspended in 50–150 µL TE.
Plasmodial DNA isolation.— – Plasmodial DNA was isolated with the OmniPrepTM Genomic DNA Extraction Kit (Geno Technology Inc., St Louis, Missouri). Cold sterile distilled water, 3.0 mL, was added to each plate and the plasmodia were harvested by scraping with a glass rod and then transferring to a 15 mL tube. The sample was centrifuged at 1650 g for 15 s. The slime layer that forms over the plasmodia was removed by aspiration. OmniPrepTM lysis buffer, 300 µL, was added to the pellet. The lysate was transferred to a 1.5 mL micro-centrifuge tube and ground with a pestle to disperse plasmodial clumps. The rest of the procedure was according to kit instructions, however the initial lysate incubation was extended to 2 h.
Probes and hybridization.— –
The mtDNA probe, Eco RI-7 (2.0 kb) was obtained from the Pan2–16 strain as described previously (Silliker et al 2002). Standard procedures were used for gel electrophoresis (Sambrook et al 1989). Bidirectional transfers of DNA from gels were according to the method of Smith and Summers (1980). Probes were labeled by the random primed method with digoxigenin and detected with an antidigoxigenin antibody conjugated to alkaline phosphatase that cleaves the chromogenic substrates, nitro-blue tetrazolium and 5-bromo-4-chloro-indoyl-phosphate (these procedures were performed as described in the Roche Molecular Biochemicals DIG Application Manual, Indianapolis, Indiana).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
To test the hypothesis that strain competency influences mitochondrial transmission reciprocal crosses were made between strains with the P mitochondrial type (Pan2–44 and Pan2–16) and those with the H mitochondrial type (Hon1–7 and Hon1–2). The term reciprocal cross in this experiment does not refer to maternal versus paternal parentage, instead it refers to pairs of crosses where the competent and precompetent parents were switched. This was accomplished by cultivating the precompetent parent at low cell densities and the competent parent to high cell densities. Hereafter these crosses will be referred to as high/low crosses.
Previously the Pan2–44 strain was the dominant mitochondrial donor (100% of the time) when mated with Hon1–7 (n = 32, in two separate trials combined) and the favored donor (85% of the time) when mated with Hon1–2 (n = 17 in two separate trials combined [Silliker et al 2002]). Our hypothesis would be supported if we saw an increased inheritance of the H mitochondrial type when the Honduran strains were the competent parent and the Panama strains were the precompetent parent at the time of combining the cells. Instead the P mitochondrial type was transmitted 100% of the time when Pan2–44 was mated with Hon1–7 regardless of the competent parent at the time of mating (TABLE I). This is consistent with previous crosses between the same parents crossed at the stationary phase. The results for the high/low crosses with Pan2–44 and Hon1–2 show favored transmittance of the H mitochondrial type regardless of the mating competence of the parents at the time of mating (TABLE I). In contrast to our previous results for this mating the biased inheritance now favors the H parent and biparental inheritance also was detected.
|
); in two trials only the P mitochondrial type was inherited (n = 13 combined data), but in a third trial the ratio of H : P inheritance was 8 : 6. In the current study the favored mitochondrial donor was the precompetent mating parent, the reverse of what our hypothesis predicted (TABLE I
). In addition biparental inheritance was observed in both high/low crosses.
In a previous study where stationary cultures of Pan2–16 and Hon1–2 were mated the ratio of P : H inheritance was 0 : 13 in one trial and 4 : 0 in another trial (Silliker et al 2002). In the current study the H mitochondrial type was inherited in all cases regardless of the competent parent and no biparental inheritance was observed (TABLE I).
Two other types of crosses were made parallel to the above crosses. Equal density crosses were made by combining equal numbers of compatible cells where both parents were just reaching competent cell densities and isolating individual plasmodia. In the earlier study (Silliker et al 2002) stationary cultures were mated and cell numbers were not equalized. Somatic fusion of plasmodia was allowed on the equal density cross plates after individual plasmodia had been isolated to see if this increased biparental inheritance. A comparison of the results from all three types of crosses is presented (TABLE II). (Given the completely dominant inheritance of the P mitochondrial type when Pan2–44 was mated with Hon1–7 in this study and the previous one, we did not continue with this cross.)
|
We wanted to be sure that our different crossing methodologies did not adversely affect mating efficiency, so observations were made on the density and timing of plasmodial formation in the high/low and the equal density crosses (TABLE III). Equal density crosses were not done for the Pan2–44 x Hon1–7 cross for the reasons mentioned above. There was no difference in plasmodial density between the two types of crosses within a mating pair, however plasmodial formation was delayed by about a day in the high/low crosses compared to the equal density matings.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
) the inheritance pattern for this mating changed in different trials. Because none of the other matings showed any effect of mating competence on inheritance the association in this one cross is no doubt spurious.
In devising our crossing methodology we were concerned that there might be differences in the mating inductive powers of the different strains. Nader et al 1984 observed that different strains of D. iridis varied in their inductive properties, although all strains seemed to respond similarly to the inducer. In our procedure the mating cells were suspended in growth media conditioned by the competent parent. If one of the parents was a weak producer of the inducer then the competency inducing environment might be less potent in one of the reciprocal crosses. We observed that the high/low crosses were delayed in plasmodial formation in comparison with the same equal density cross but the reciprocal crosses were delayed to the same degree. Presumably the pre-competent cells could not mate with competent cells until they had made the transition to competence. Apparently, delaying the mating of the competent parents did not affect mating efficiency since plasmodial density was comparable between high/low and equal density crosses. This suggests that once cells are induced to competency they maintain this state under our experimental conditions.
In this study the results for different trials of a mating were fairly consistent, as were the results for all three types of crosses, high/low, equal density, and somatic fusion (with the exception of the Pan2–16 x Hon1–7 high/low cross). However if cross type is ignored and the combined data for each mating is compared to the previous study (Silliker 2002) using these strains, several differences emerge. Only the results of the Pan2–44 x Hon1–7 mating are completely consistent with the previous study (in both studies the P mitochondrial type was uniformly inherited). For both the Pan2–44 x Hon1–2 and the Pan2–16 x Hon1–7 matings the favored mitochondrial donor changed from the P-parent in the previous study to the H-parent in the current study. For the Pan2–16 x Hon1–2 mating, the H-parent was the favored donor in the previous study, in this study only the H mitochondrial type was transmitted. The combined data support the contention that for certain matings the observed mitochondrial inheritance pattern is highly conditional, although we have not determined the factors that influence the pattern.
Ross et al 1973 observed that mating efficiency was strongly affected by cultural history; recently excysted amoebae gave variable mating results compared to crosses made with amoebae that had been actively growing through several transfers. Our mating cultures were maintained as actively growing cultures. We have found that Pan2–16 mitochondria contain subgenomic mitochondrial DNA molecules that change in their proportion over time (Silliker 2003). Whether the composition of these subgenomic molecules affects mitochondrial transmission remains to be seen. These small molecules might improve or decrease mitochondrial function and therefore give the mitochondria a selective advantage or disadvantage, but that would presumably be a slow process. The establishment of uniparental mitochondrial populations in plasmodia after mating is a rapid process (Silliker et al 2002). In P. polycephalum Moriyama and Kawano (2003) have shown rapid, selective degradation of one parental mitochondrial type after mating.
The observations of Moriyama and Kawano 2003 also suggest that there is a window of time after mating when the resident mitochondrial type is established. They argue that, in P. polycephalum, biparental inheritance results from a failure to completely digest one of the parental mitochondrial types shortly after mating. In their model, once a mitochondrial type escapes the winnowing process it is free to proliferate. If the same mechanism were operating in D. iridis we would expect to see a greater incidence of biparental inheritance in the somatic fusion crosses where individuals with potentially different mitochondrial types could fuse over time, compared to the crosses where individual plasmodia were isolated for analysis, however this was not observed.
Considering only the life cycle of D. iridis, one might expect a high level of biparental mitochondrial inheritance in plasmodia, yet uniparental inheritance is observed more frequently. Given the strong tendency toward uniparental inheritance, which requires an active selection process, it seems remarkable that the donating parent can vary in some matings. The genetic and environmental determinants of the observed patterns remain elusive.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
1 Corresponding author. E-mail: msillike{at}depaul.edu
|
LITERATURE CITED |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIAL AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
———. 2001. The inheritance of genes in mitochondria and chloroplasts: laws, mechanisms, and models. Annu Rev Genet 35:125–148.[CrossRef][Medline]
Clark J, Collins OR, Tang HC. 1991. Didymium iridis mating systems: partial compatibility between mating series. Mycologia 83:210–213.[CrossRef]
Collins OR. 1963. Multiple alleles at the incompatibility locus in the myxomycete Didymium iridis. Amer J Bot 50:477–480.[CrossRef]
———, Betterley DA. 1982. Didymium iridis in past and future research. In: Aldrich HC, Daniels JW, eds. Cell biology of Physarum and Didymium. New York, New York: Academic Press. p 27–57.
Eberhard WG. 1980. Intraorganism competition involving eukaryotic organelles. Annals New York Acad of Sci p 44–50.
Gillham NW. 1994. Organelle genes and genomes. New York, New York: Oxford University Press. 424 p.
Kawano S, Anderson RW, Nanba T. 1987. Polymorphism and uniparental inheritance of mitochondrial DNA in Physarum polycephalum. J Gen Micro 133:3175–3182.
———, Kuroiwa T. 1989. Transmission pattern of mitochondrial DNA during plasmodium formation in Physarum polycephalum. J Gen Micro 135:1559–1566.
Meland S, Johansen S, Johansen T, Haugli K, Haugli F. 1991. Rapid disappearance of one parental mitochondrial genotype after isogamous mating in the myxomycete Physarum polycephalum. Curr Genet 19:55–60.[CrossRef][Medline]
Moriyama Y, Kawano S. 2003. Rapid, selective digestion of mitochondrial DNA in accordance with the matA locus hierarchy of multiallelic mating types in the mitochondrial inheritance of Physarum polycephalum. Genetics 164:963–975.
Nader WF, Shipley GL, Huettermann A, Holt CE. 1984. Analysis of an inducer of the ameobal-plasmodial transition in the myxomycetes Didymium iridis and Physarum polycephalum. Devel Biol 103:504–510.[CrossRef][Medline]
Olive LS. 1975. The Mycetozoans. New York, New York: Academic Press. 293 p.
Partridge L, Hurst LD. 1998. Sex and Conflict. Science 281:2003–2008.
Ross IK, Shipley GL, Cummings RJ. 1973. Sexual and somatic cell fusions in the heterothallic slime mould Didymium iridis 1 Fusion assay, fusion kinetics and cultural parameters. Microbios 7:119–164.
Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular Cloning: a laboratory manual. 2nd ed. Cold Spring Harbor, New York: Cold Spring Harbor Press.
Shipley GL, Holt CE. 1982. Cell fusion competence and its induction in Physarum polycephalum and Didymium iridis. Devel Biol 90:110–117.[CrossRef][Medline]
Silliker ME, Collins OR. 1988. Non-mendelian inheritance of mitochondrial DNA and ribosomal DNA in the myxomycete, Didymium iridis. Mol Gen Genet 213:370–378.[CrossRef][Medline]
———, Gong T, Collins OR. 1988. Spore-to-spore cultivation of Didymium iridis on heat killed bacteria. Mycologia 80:151–156.[CrossRef]
———, Liles JL, Monroe JA. 2002. Patterns of mitochondrial inheritance in the myxogastrid, Didymium iridis. Mycologia 94:939–946.
———, Tiu AP, Smith BJ. 2003. Small mitochondrial DNA molecules of the plasmodial slime mold Didymium iridis contain mitochondrial genes sequences. Inoculum 54:45 abstract.
Smith GE, Summers MD. 1980. The bi-directional transfer of DNA to nitrocellulose or diabenzylomethyl-paper. Anal Biochem 109:123–129.[CrossRef][Medline]
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
