Mitochondrial Genetic Analysis by Zygote Cell Lineages in SACCHAROMYCES CEREVISIAE

Yeast strains were constructed carrying multiple mitochondrial markers conferring resistance to the inhibitors erythromycin, chloramphenicol, paromomycin and oligomycin. A pedigree analysis of two crosses was made by micromanipulating buds from zygotes. The first few daughter buds isolated from the zygotes sometimes gave rise to diploid clones which had a mixture of mitochondrial types. All possible classes of mitochondrial parental and recombinant types were found although they never appeared all together as the progeny from a single zygote. It was inferred that multiple recombination events took place in zygotes and in some of the buds derived from them. After removal of the first four or so daughter buds, subsequent buds from the zygote carried one mitochondrial type only. In cross I in which three markers were analyzed this was most frequently one of the parental types. In cross II (involving four mitochondrial markers) the later buds from the zygotes were frequently of recombinant mitochondrial type.     

Vegetative segregation of mitochondria in yeast: Estimating parameters using a random model

Summary Yeast zygotes which are heteroplasmic for mitochondrial genes reproduce vegetatively to form clones of diploid progeny which are homoplasmic. This vegetative segregation of mitochondrial genes has been interpreted in terms of a random distribution of mitochondria or mitochondrial genomes between mother and bud at cell division. We have developed equations which permit calculation of the number of segregating units in the zygote and the number of those units which enter the bud, assuming that segregation of the units is genetically random and numerically variable or equal. Use of the equations requires data from partial pedigree analyses: we isolate zygotes, separate the first bud, then determine the frequency of mitochondrial alleles among the progeny of mother cells whose first buds were homoplasmic. Application of this method to data from five crosses suggests that most zygotes have a small number of segregating units (usually less than a dozen) and only one or two enter the first bud. Analysis of the frequency of buds which are nearly but not quite homoplasmic indicates that the segregating units may be mitochondria or portions thereof which include many mitochondrial genomes, all the genomes in a unit being genetically identical in most but not all cases. These results are compatible with, but do not prove, the hypothesis of random vegetative segregation of mitochondria.     

The mitochondrial genome of wild-type yeast cells. VII. Recombination in crosses.

Two Saccharomyces cerevisiae wild-type strains were crossed, and 26 diploid clones were obtained from (1) mass mating; (2) individual buds in zygote lineages; (3) individual zygotes. The mitochondrial DNAs from these diploids were investigated in their recombination and segregation by analyzing their restriction fragment patterns.Recombinant mitochondrial genomes were present in 75% of the diploid clones. Such recombinant genomes had unit sizes different from, yet within ± 5% of, the parental ones and showed EcoRI and HindII + III fragment patterns of parental types, two strong indications that both the gene complement and the gene order were very largely preserved in the progeny.Fragment patterns produced by HpaII and HaeIII were characterized by (1) fragments originating from the DNAs of both parents; and (2) new fragments, namely fragments absent in either parent. The new fragments appear to arise from unequal crossing-over events occurring in the spacers of allelic parental genetic units and usually have preferential localizations in the genome.These results provide the first evidence for physical recombinations of mitochondrial DNA in crosses of wild-type yeast cells, indicate that recombination is very frequent in crosses, and shed some light on mitochondrial segregation. They also have interesting implications for recombination phenomena in interspersed systems of unique and repetitive nucleotide sequences.     

Patterns of mitochondrial sorting in yeast zygotes.

Inheritance of mitochondrial DNA (mtDNA) in Saccharomyces cerevisiae is usually biparental. Pedigree studies of zygotic first buds indicate limited mixing of wild-type (p+) parental mtDNAs: end buds are frequently homoplasmic for one parental mtDNA, while heteroplasmic and recombinant progeny usually arise from medial buds. In crosses involving certain petites, however, mitochondrial inheritance can be uniparental. In this study we show that mitochondrial sorting can be influenced by the parental mtDNAs and have identified intermediates in the process. In crosses where mtDNA mixing is limited and one parent is prelabeled with the matrix enzyme citrate synthase 1 (CS1), the protein freely equilibrates throughout the zygote before the first bud has matured. Furthermore, if one parent is p0 (lacking mtDNA), mtDNA from the p+ parent can also equilibrate; intracellular movement of mtDNA is unhindered in this case. Surprisingly, in zygotes from a p0 CS1+ x p+ CS1- cross, CS1 is quantitatively translocated to the p+ end of the zygote before mtDNA movement; subsequently, both components equilibrate throughout the cell. This initial vectorial transfer does not require respiratory function in the p+ parent, although it does not occur if that parent is p-. Mouse dihydrofolate reductase (DHFR) present in the mitochondrial matrix can also be vectorially translocated, indicating that the process is general. Our data suggest that in zygotes mtDNA movement may be separately controlled from the movement of bulk matrix constituents.     

Intracellular Population Genetics: Evidence for Random Drift of Mitochondrial Allele Frequencies in SACCHAROMYCES CEREVISIAE and SCHIZOSACCHAROMYCES POMBE

We report evidence for random drift of mitochondrial allele frequencies in zygote clones of Saccharomyces cerevisiae and Schizosaccharomyces pombe. Monofactorial and bifactorial crosses were done, using strains resistant or sensitive to erythromycin (alleles E^R, E^S), oligomycin (O^R, O^S), or diuron (D^R, D^S). The frequencies of resistant and sensitive cells (and thus the frequencies of the resistant and sensitive alleles) were determined for each of a number of clones of diploid cells arising from individual zygotes. Allele frequencies were extremely variable among these zygote clones; some clones were "uniparental," with mitochondrial alleles from only one parent present. These observations suggest random drift of the allele frequencies in the population of mitochondrial genes within an individual zygote and its diploid progeny. Drift would cease when all the cells in a clone become homoplasmic, due to segregation of the mitochondrial genomes during vegetative cell divisions. To test this, we delayed cell division (and hence segregation) for varying times by starving zygotes in order to give drift more time to operate. As predicted, delaying cell division resulted in an increase in the variance of allele frequencies among the zygote clones and an increase in the proportion of uniparental zygote clones. The changes in form of the allele frequency distributions resembled those seen during random drift in finite Mendelian populations. In bifactorial crosses, genotypes as well as individual alleles were fixed or lost in some zygote clones. However, the mean recombination frequency for a large number of clones did not increase when cell division was delayed. Several possible molecular mechanisms for intracellular random drift are discussed.     

The biogenesis of mitochondria 26

Summary The vegetative segregation of parental and recombinant mitochondrial (cytoplasmic) genomes of Saccharomyces cerevisiae were compared by experiments involving the micromanipulation of early zygotic buds. Recombinant mitochondrial genomes are formed rapidly upon zygote formation and initial zygote buds are frequently composed of varying proportions of recombinant and parental type gnomes, which then all segregate in a similar fashion. Evidence suggesting that some formation of recombinant genomes can continue in the early zygote progeny is presented, but the possibility that recombination is restricted to the zygote has not been excluded. Polarity phenomena appear to be determined by the mechanism of the mitochondrial recombination events rather than by the mechanism of distribution of recombinant genomes to zygote progeny.     

Kinetic and segregational analysis of mitochondrial DNA recombination in yeast

A pair of yeast strains of opposite mating type was constructed to contain polymorphisms at three loci on the mitochondrial genome--the 21 S rRNA gene, var1, and cob--such that parental and recombinant forms of these genes could be easily detected by Southern blot analysis. These polymorphisms were used to measure in a single cross gene conversions at the 21 S rRNA and var1 loci and a reciprocal recombination at cob. For all three loci, recombination initiates at about the same time, 4 to 6 h after mixing cells, and increases with similar kinetics over a 24-h period. The segregation of parental and recombinant forms of these genes was then followed by pedigree analysis. The results, which show a high variance in the distribution of parental and recombinant forms of all three genes in cells derived from both the first bud and the mother zygote, are consistent with the segregation of a small number of mitochondrial DNA molecules from the zygote to diploid buds. Based on these results and previous experiments of this type, a limited "zone of mixing" of parental mitochondrial DNA molecules probably exists in the zygote. The extent of sampling from this zone, together with the intrinsic properties of the recombination events themselves, is likely to determine the observed pattern of recombination of mitochondrial DNA sequences at the population level.     

Transmission, segregation, and recombination of mitochondrial genomes in zygote clones and protoplast fusion clones of yeast

Summary The average transmission-and recombination frequencies of mitochondrial markers are similar in diploid clones derived from zygotes or from fusion of haploid protoplasts of identical mating type. The transmissional patterns of mitochondrial markers in individual fusion-or zygote-clones, however, are very different. The time needed for regeneration of cell walls of fused protoplasts is found to be mainly responsible for this difference, since delay of first cell division in zygotes leads to similar results.     

Uniparental Inheritance of Mitochondrial Genes in Yeast: Dependence on Input Bias of Mitochondrial DNA and Preliminary Investigations of the Mechanism

In Saccharomyces cerevisiae, previous studies on the inheritance of mitochondrial genes controlling antibiotic resistance have shown that some crosses produce a substantial number of uniparental zygotes, which transmit to their diploid progeny mitochondrial alleles from only one parent. In this paper, we show that uniparental zygotes are formed especially when one parent (majority parent) contributes substantially more mitochondrial DNA molecules to the zygote than does the other (minority) parent. Cellular contents of mitochondrial DNA (mtDNA) are increased in these experiments by treatment with cycloheximide, alpha-factor, or the uvsp5 nuclear mutation. In such a biased cross, some zygotes are uniparental for mitochondrial alleles from the majority parent, and the frequency of such zygotes increases with increasing bias. In two- and three-factor crosses the cap1, ery1, and oli1 loci behave coordinately, rather than independently; minority markers tend to be transmitted or lost as a unit, suggesting that the uniparental mechanism acts on entire mtDNA molecules rather than on individual loci. This rules out the possibility that uniparental inheritance can be explained by the conversion of minority markers to the majority alleles during recombination. Exceptions to the coordinate behavior of different loci can be explained by marker rescue via recombination. Uniparental inheritance is largely independent of the position of buds on the zygote. We conclude that it is due to the failure of minority markers to replicate in some zygotes, possibly involving the rapid enzymatic destruction of such markers. We have considered two general classes of mechanisms: (1) random selection of molecules for replication, as for example by competition for replicating sites on a membrane; and (2) differential marking of mtDNA molecules in the two parents, possibly by modification enzymes, followed by a mechanism that "counts" molecules and replicates only the majority type. These classes of models are distinguished genetically by the fact that the first predicts that the output frequency of a given allele among the progeny of a large number of zygotes will approximately equal the average input frequency of that allele, while the second class predicts that any input bias will be amplified in the output. The data suggest that bias amplification does occur. We hypothesize that maternal inheritance of mitochondrial or chloroplast genes in many organisms may depend upon a biased input of organelle DNA molecules, which usually favors the maternal parent, followed by failure of the minority (paternal) molecules to replicate in many or all zygotes.     

Extrachromosomal inheritance in Schizosaccharomyces pombe

Summary Primary and secondary spore clones were analyzed from two- and three-factor crosses involving the mitochondrial markers conferring resistance to antimycin (A ^R ), chloramphenicol (C ^R ), and erythromycin (E ^R ). As in zygote clones (Seitz-Mayr et al., 1978), transmission of markers is higher in two-factor trans-crosses than in cis-crosses. Except transmission of C ^R in the cross A ^R C ^R E ^R xA ^S C ^{S E S} , no significant differences between cis- and trans-configuration were observed in three-factor crosses. In contrast to zygote clones, in spore clones transmission rates of the two or three markers in a given cross are roughly equal. 18 out of 20 secondary spore clones of different mitochondrial phenotypes appeared to be homoplasmic, whereas 2 still continued to segregate. One of these spore isolates was analyzed, and segregation was found to continue for more than 150 generations after spore germination. Whereas up to more than 80% of zygote clones in certain crosses were uniform, only 2 out of 91 tetrads were uniform, i.e. all four spores were homoplasmic for the same mitochondrial genotype. Presence or absence of recombinant mitochondrial phenytypes among secondary spore clones from tetrads indicated, whether, cytoplasmic mixing had occurred in the original zygote or not. Within an ascus, the number of spores containing recombinant genotype(s) is a direct measure for the extent of cytoplasmic mixing in the zygote. In 82 tetrads analyzed, the number of tetrads with 0, 1, 2, 3, and 4 spores containing recombinant genotype(s) were 25, 37, 14, 5, and 1, respectively. In conclusion, the extent of cytoplasmic mixing at the cell stage before forespore membrane formation is highly variable.     

The non-reciprocality of organelle gene recombination in Chlamydomonas reinhardtii and Saccharomyces cerevisiae: some new observations and a restatement of some old problems.

Summary Organelle recombinant genotype frequencies, derived from analysis of individual mitotic zygote clones of Chlamydomonas reinhardtii and Saccharomyces cerevisiae, were subjected to two types of statistical tests in an attempt to detect the occurrence of reciprocal recombination: (i) calculation of correlation coefficients for the frequencies of two recombinant genotypes (reciprocal or non-reciprocal pairs) within individual zygote clones, and (ii) application of the chi-square test for independence to the frequencies of zygotes yielding one or the other, neither, or both of a given recombinant pair.Applying test (i), the strongest correlations are found for non-reciprocal rather than reciprocal pairs. When the data are analyzed by method (ii), some reciprocal as well as non-reciprocal pairs appear to be produced concurrently in zygote clones. However, such deviations from independence are greatest for non-reciprocal pairs. These tests yield comparable results for yeast mitochondrial and Chlamydomonas chloroplast gene recombination, and provide no convincing evidence for reciprocal genetic exchange. Explanations for the observed lack of reciprocality are discussed with reference both to our present understanding of the molecular events responsible for genetic recombination, and to the problems which may be unique to the analysis of organelle gene recombination.     

Chloroplast genetics of chlamydomonas. I. Allelic segregation ratios

This paper presents allelic segregation data from a series of 16 crosses segregated for nuclear and chloroplast genes. By means of pedigree analysis, segregants of chloroplast markers occurring in the zygote have been distinguished from those occurring in zoospore clones. The genes ac1, ac2, and tm1 showed little if any deviation from 1:1 either in zygotic segregation or in zoospore clones. The genes sm2, ery, and spc showed a significant excess of the allele from the mt (+) parent in zygotes. However, in zoospores, mt( +) excess was seen only when that allele was the mutant (resistant) form but not when it was wild type (sensitive).These results show that the extent of preferential segregation differs in zygotes and in zoospores, and that preferential segregation is influenced by map location and by allele specificity. A comparison of progeny from zygotes mated after 0, 15', 30', and 50' UV irradiation of the mt(+) gametes demonstrated the lack of an effect of UV upon allelic segregation ratios. In total, these results exclude the multi-copy model of chloroplast genome segregation suggested by Gillham, Boynton and Lee (1974) and support the diploid model we have previously proposed (Sager and Ramanis 1968, 1970; Sager 1972).     

Estimation of the position and effect of a lethal factor locus on a molecular marker linkage map

In the mapping of DNA markers the distortion of segregation of marker genotypes is often observed, which may be caused by a lethal factor acting in filial generations derived from distant crosses. A method is presented for estimating the recombination values between a lethal factor locus and neighboring molecular markers, and the relative viability or fertilization ability of gametes or zygotes affected by the lethal factor in an F₂ population using the maximum likelihood method and the expectation conditional maximization (ECM) algorithm. Three selection models of gamete or zygote were considered, and the most likely one was determined by goodness of fit of the observed frequency of the phenotypes to the expected ones under the models. The method was applied to segregation data of molecular markers of an F₂ population consisting of 144 individuals derived from a cross between an Indica and a Japonica rice variety. The presence of a lethal factor locus (L) located on chromosome III that caused partial gametic selection in both the male and female sides was suggested. The locus L was tightly linked to RFLP marker number 23 of the RFLP linkage map of Saito et al. (1991a), and the fertilization chance of a male or female gamete possessing the lethal factor was, on average, 41.5% of that of the normal gamete.     

Distribution of mitochondrially inherited drug-resistance genes to tetrads from young zygotes in yeast

Summary Pairs of strains of opposite mating type were isolated from a strain of Saccharomyces cerevisiae. From these isogenic strains, mitochondrially inherited resistant mutants to antimycin A and erythromycin were isolated. By using the two resistance genes as mitochondrial markers, it was proposed that the distribution of the mitochondrial genomes from zygotes to tetrads seemed not to be random but the genomes from either a or parent would be selected with approximately equal frequencies after zygote formation and subsequently distributed uniparentally to meiotic products.     

Transmission and recombination of mitochondrial genes in Saccharomyces cerevisiae after protoplast fusion

Summary Protoplasts of auxotrophic strains of Saccharomyces cerevisiae of opposite and identical mating types carrying different mitochondrial drug-resistance markers, with both homosexual and heterosexual mitochondrial backgrounds, were induced to fuse by polyethylene glycol. After selective regeneration of prototrophic fusion products, the transmission and recombination frequencies of mitochondrial genes in populations of cells were determined and compared with those obtained in mating processes. The frequencies obtained in the fusion experiments proved very similar to those found in the zygote clones. The behavior of mitochondrial genes was apparently affected neither by nuclear mating type background nor by the method of transfer of mitochondrial genomes (i.e., protoplast fusion or mating), making possible mitochondrial genetic studies by protoplast fusion irrespective of the mating type barrier of yeast strains.     

Effect of antibiotics on the transmission of mitochondrial factors in Saccharomyces cerevisiae

Summary Transmission of mitochondrial factors was studied in zygote clones, random diploids, and zygote cell lineages. An asymmetrical distribution of both parental and recombinant classes was consistently seen. Genetic analysis indicated that a nuclear factor was controlling this pattern. Treatment of young zygotes with either cycloheximide or thiolutin for 90 minutes destroyed the pattern and randomized the distribution of parental and recombinant classes. Inhibition of mitochondrial protein synthesis with antibacterial antibiotics had no detectable effect on the pattern of distribution of mitochondrial markers.     

Cytoplasmic inheritance in Saccharomyces cerevisiae: Comparison of zygotic mitochondrial inheritance patterns

Summary Mitochondrial movements in Saccharomyces cerevisiae (Sc) zygotes were monitored with phase-contrast microscopy and compared to known mitochondrial inheritance systems. The mitochondria of Sc were convincingly identified by integrated use of phase-contrast, cytochemical and electron microscopic observations. Mitochondria in Sc appear to move by saltatory jumps, which appear to be oriented towards movement of mitochondria into developing buds. Tracking of mitochondria of different genotypes was made possible by positive identification of each mitochondrial population before zygosis, and by the low degree of mixing (<10%) of mitochondrial populations before first bud septation.A grande by grande cross demonstrated equal numbers of mitochondria from each haploid moving into the first zygotic bud. A grande by neutral petite cross gave a 2:1 ratio of grande to petite mitochondria. However, a grande by suppressive petite cross gave equal numbers of grande and petite mitochondria. Using drug resistance systems, a comparison was made of highly biased (97%) and moderately biased (71%) chloramphenicol resistant inheritance patterns. In both cases, the ratios of drug resistant to sensitive mitochondria were 1:1. When numbers of mitochondria moving into an individual bud were compared to the phenotypic content of the clone of that bud, no model could be constructed which could predict the latter from the former. The data indicate (with the exception of the neutral petite by grande cross) that the numbers of each mitochondrial type inserted into the first zygotic bud are equal, regardless of the degree of asymmetry of inheritance of mitochondrial markers.     

Genetic mapping of mitochondrial markers by recombinational analysis in Chlamydomonas reinhardtii

The mitochondrial genome of Chlamydomonas reinhardtii is a 15.8 kb linear DNA molecule present in multiple copies. In crosses, the meiotic products only inherit the mitochondrial genome of the mating type minus (paternal) parent. In contrast mitotic zygotes transmit maternal and paternal mitochondrial DNA copies to their diploid progeny and recombinational events between molecules of both origins frequently occur. Six mitochondrial mutants unable to grow in the dark (dk^– mutants) were crossed in various combinations and the percentages of wild-type dk⁺ recombinants were determined in mitotic zygotes when all progeny cells had become homoplasmic for the mitochondrial genome. In crosses between strains mutated in the COB (apocytochrome ) gene and strains mutated in the COX1 (subunit 1 of cytochrome oxidase) gene, the frequency of recombination was 13.7% (± 3.2%). The corresponding physical distance between the mutation sites was 4.3 kb. In crosses between strains carrying mutations separated by about 20 bp, a recombinational frequency of 0.04% (± 0.02%) was found. Two other mutants not yet characterized at the molecular level were also used for recombinational studies. From these data, a linear genetic map of the mitochondrial genome could be drawn. This map is consistent with the positions of the mutation sites on the mitochondrial DNA molecule and thereby validates the method used to generate the map. The frequency of recombination per physical distance unit (3.2% ± 0.7% per kilobase) is compared with those obtained for other organellar genomes in yeasts and Chlamydomonas.     

Mitochondrial fusion and inheritance of the mitochondrial genome

Although maternal or uniparental inheritance of mitochondrial genomes is a general rule, biparental inheritance is sometimes observed in protists and fungi, including yeasts. In yeast, recombination occurs between the mitochondrial genomes inherited from both parents. Mitochondrial fusion observed in yeast zygotes is thought to set up a space for DNA recombination. In the last decade, a universal mitochondrial fusion mechanism has been uncovered, using yeast as a model. On the other hand, an alternative mitochondrial fusion mechanism has been identified in the true slime mold Physarum polycephalum. A specific mitochondrial plasmid, mF, has been detected as the genetic material that causes mitochondrial fusion in P. polycephalum. Without mF, fusion of the mitochondria is not observed throughout the life cycle, suggesting that Physarum has no constitutive mitochondrial fusion mechanism. Conversely, mitochondria fuse in zygotes and during sporulation with mF. The complete mF sequence suggests that one gene, ORF640, encodes a fusogen for Physarum mitochondria. Although in general, mitochondria are inherited uniparentally, biparental inheritance occurs with specific sexual crossing in P. polycephalum. An analysis of the transmission of mitochondrial genomes has shown that recombinations between two parental mitochondrial genomes require mitochondrial fusion, mediated by mF. Physarum is a unique organism for studying mitochondrial fusion.     

Frequency distributions for chloroplast genes in Chlamydomonas zygote clones: Evidence for random drift

Mitochondria and chloroplasts of eucaryotic cells contain populations of DNA molecules. In certain cases, e.g., the chloroplasts of Chlamydomonas reinhardtii and the mitochondria of Saccharomyces cerevisiae, organelles contributed by the two parents are known to fuse in the zygote, creating a single population of DNA molecules. In a cross, this population will include molecules of both parental genotypes. There is reason to suspect that organelle DNA molecules in this population are selected randomly for replication and recombination. This would result in random changes in the frequency of a particular allele or genotype within the organelle gene pool of a single zygote and also within its clone of progeny cells. A given gene frequency would increase in some zygote clones and decrease in others, analogous to random drift of gene frequencies in small Mendelian populations. To test this, we have examined the distribution of chloroplast gene frequencies among the zygote clones produced in each of a number of crosses of Chlamydomonas. These distributions are typically U or L shaped as predicted by the random drift hypothesis. They include uniparental zygote clones, in which a chloroplast allele from one parent has been fixed (frequency 100%) and the alternative allele from the other parent has been lost (frequency 0%). Among the remaining (biparental) zygote clones, there is a linear distribution of allele frequencies, showing a great increase in variance over the input frequencies. In these experiments both biparental and uniparental zygotes show a bias favoring chloroplast alleles from the mt⁺ (maternal) parent, and there is no statistically significant mode at the allele frequency of 0.5 corresponding to the equal input of alleles from the maternal and paternal (mt^?) parents. The observed distributions support the hypothesis that both uniparental inheritance and the high variance of allele frequencies among zygote clones are due to random drift of allele frequencies, coupled with a directional force which favors fixation of the maternal allele. In addition, statistical analysis of the data shows a strong but incomplete tendency for linked chloroplast markers to be fixed or lost together in uniparental zygotes. Possible cellular and molecular mechanisms for these observations are discussed.