The inheritance of genes in mitochondria and chloroplasts: laws, mechanisms, and models.

The inheritance of mitochondrial and chloroplast genes differs from that of nuclear genes in showing vegetative segregation, uniparental inheritance, intracellular selection, and reduced recombination. Vegetative segregation and some cases of uniparental inheritance are due to stochastic replication and partitioning of organelle genomes. The rate and pattern of vegetative segregation depend partly on the numbers of genomes and of organelles per cell, but more importantly on the extent to which genomes are shared between organelles, their distribution in the cell, the variance in number of replications per molecule, and the variance in numerical and genotypic partitioning of organelles and genomes. Most of these parameters are unknown for most organisms, but a simple binomial probability model using the effective number of genomes is a useful substitute. Studies using new cytological, molecular, and genetic methods are shedding some light on the processes involved in segregation, and also on the mechanisms of intracellular selection and uniparental inheritance in mammals. But significant issues remain unresolved, notably about the extent of paternal transmission and mitochondrial fusion in mammals.

Accelerated evolution of functional plastid rRNA and elongation factor genes due to reduced protein synthetic load after the loss of photosynthesis in the chlorophyte alga Polytoma.

Polytoma obtusum and Polytoma uvella are members of a clade of nonphotosynthetic chlorophyte algae closely related to Chlamydomonas humicola and other photosynthetic members of the Chlamydomonadaceae. Descended from a nonphotosynthetic mutant, these obligate heterotrophs retain a plastid (leucoplast) with a functional protein synthetic system, and a plastid genome (lpDNA) with functional genes encoding proteins required for transcription and translation. Comparative studies of the evolution of genes in chloroplasts and leucoplasts can identify modes of selection acting on the plastid genome. Two plastid genes--rrn16, encoding the plastid small-subunit rRNA, and tufA, encoding elongation factor Tu--retain their functions in protein synthesis after the loss of photosynthesis in two nonphotosynthetic Polytoma clades but show a substantially accelerated rate of base substitution in the P. uvella clade. The accelerated evolution of tufA is due, at least partly, to relaxed codon bias favoring codons that can be read without wobble, mainly in three amino acids. Selection for these codons may be relaxed because leucoplasts are required to synthesize fewer protein molecules per unit time than are chloroplasts (reduced protein synthetic load) and thus require a lower rate of synthesis of elongation factor Tu. Relaxed selection due to a lower protein synthetic load is also a plausible explanation for the accelerated rate of evolution of rrn16, but the available data are insufficient to test the hypothesis for this gene. The tufA and rrn16 genes in Polytoma oviforme, the sole member of a second nonphotosynthetic clade, are also functional but show no sign of relaxed selection.

Heterozygosity, heteromorphy, and phylogenetic trees in asexual eukaryotes.

Little attention has been paid to the consequences of long-term asexual reproduction for sequence evolution in diploid or polyploid eukaryotic organisms. Some elementary theory shows that the amount of neutral sequence divergence between two alleles of a protein-coding gene in an asexual individual will be greater than that in a sexual species by a factor of 2tu, where t is the number of generations since sexual reproduction was lost and u is the mutation rate per generation in the asexual lineage. Phylogenetic trees based on only one allele from each of two or more species will show incorrect divergence times and, more often than not, incorrect topologies. This allele sequence divergence can be stopped temporarily by mitotic gene conversion, mitotic crossing-over, or ploidy reduction. If these convergence events are rare, ancient asexual lineages can be recognized by their high allele sequence divergence. At intermediate frequencies of convergence events, it will be impossible to reconstruct the correct phylogeny of an asexual clade from the sequences of protein coding genes. Convergence may be limited by allele sequence divergence and heterozygous chromosomal rearrangements which reduce the homology needed for recombination and result in aneuploidy after crossing-over or ploidy cycles.

Uniparental inheritance of mitochondrial and chloroplast genes: mechanisms and evolution.

In nearly all eukaryotes, at least some individuals inherit mitochondrial and chloroplast genes from only one parent. There is no single mechanism of uniparental inheritance: organelle gene inheritance is blocked by a variety of mechanisms and at different stages of reproduction in different species. Frequent changes in the pattern of organelle gene inheritance during evolution suggest that it is subject to varying selective pressures. Organelle genes often fail to recombine even when inherited biparentally; consequently, their inheritance is asexual. Sexual reproduction is apparently less important for genes in organelles than for nuclear genes, probably because there are fewer of them. As a result organelle sex can be lost because of selection for special reproductive features such as oogamy or because uniparental inheritance reduces the spread of cytoplasmic parasites and selfish organelle DNA.

Biased gene conversion, copy number, and apparent mutation rate differences within chloroplast and bacterial genomes.

We investigate the possibility that differences between synonymous substitution rates of organelle and bacterial genes differing only in copy number may be due to conversion bias. We find that the rather large observed difference in the synonymous rates between genes in the single copy and inverted-repeat regions of chloroplasts can be accounted for by a very small bias against new mutants. More generally, differences in the within-organelle fixation probability result in different apparent mutation rates as measured by the expected rate of appearance of cells homoplasmic for new mutants. Thus, differences in intracellular population parameters rather than molecular mechanisms can account for some variation in the apparent mutation rates of organelle genes, and possibly in other systems with variable numbers of gene copies. On the other hand, our analysis suggests that conversion bias is not a likely explanation for relatively low mutation rates observed near the replication origin of bacterial chromosomes.

Organelle gene diversity under migration, mutation, and drift: equilibrium expectations, approach to equilibrium, effects of heteroplasmic cells, and comparison to nuclear genes.

We developed stochastic population genetic theory for mitochondrial and chloroplast genes, using an infinite alleles model appropriate for molecular genetic data. We considered the effects of mutation, random drift, and migration in a finite island model on selectively neutral alleles. Recurrence equations were obtained for the expectation of gene diversities within zygotes, within colonies, and between colonies. The variables are number and sizes of colonies, migration rates, sex ratios, degree of paternal transmission, number of germ line cell divisions, effective number of segregating organelle genomes, and mutation rate. Computer solutions of the recurrence equations were used to study the approach to equilibrium. Gene diversities equilibrate slowly, while GST, used to measure population subdivision, equilibrates rapidly. Approximate equilibrium equations for gene diversities and GST can be obtained by substituting Neo and me, simple functions of the numbers of breeding or migrating males and females and of the degree of paternal transmission, for the effective numbers of genes and migration rates in the corresponding equations for nuclear genes. The approximate equations are not valid when the diversity within individuals is large compared to that between individuals, as is often true for the D-loop of animal mtDNA. We used the exact equations to verify that organelle genes often show more subdivision than nuclear genes; however, we also identified the range of breeding and migrating sex ratios for which population subdivision is greater for nuclear genes. Finally, we show that gene diversities are higher for nuclei than for organelles over a larger range of sex ratios in a subdivided population than in a panmictic population.

Effects of linkage on rates of molecular evolution.

When an advantageous mutation is fixed in a population by selection, a closely linked selectively neutral or mildly detrimental mutation may "hitchhike" to fixation along with it. It has been suggested that hitchhiking might increase the rate of molecular evolution. Computer simulations and a mathematical argument show that complete linkage to either advantageous or deleterious mutations does not affect the substitution of selectively neutral mutations. However, the simulations show that linkage to selected background mutations decreases the rate of fixation of advantageous mutations and increases the rate of fixation of detrimental mutations. This is true whether the linked background mutations are advantageous or detrimental, and it verifies and extends previous observations that linkage tends to reduce the effects of selection on evolution. These results can be interpreted in terms of the Hill-Robertson effect: a locus linked to another locus under selection experiences a reduction in effective population size. The interpretation of differences in evolutionary rates between different genomes or different regions of a genome may be confounded by the effects of strong linkage and selection. Recombination is expected to reduce the overall rate of molecular evolution while enhancing the rate of adaptive evolution.

Chloroplast DNA diversity is low in a wild plant, Lupinus texensis.

Chloroplast DNA diversity was measured in an annual flowering plant, Lupinus texensis. Individual plants were collected from 21 local populations throughout the range of the species in Texas. Chloroplast DNA was isolated separately from each plant and digested with seven restriction enzymes. The most common form of the 150-kilobase-pair genome was cut at 134 sites, so that about 0.5% of the base pairs in the genome were sampled. Of the 100 plants examined, 88 had identical restriction fragment patterns. Three variant forms were found in different local populations. Two, represented in single plants, differed from wild type in the presence or absence of single restriction sites. The third variant was fixed in one of the local populations; it had lost a restriction site and also had a deletion of approximately equal to 100 base pairs. The data suggest that chloroplast DNA in this plant is much less polymorphic than mitochondrial DNA from animals and is probably less polymorphic than nuclear genes in the same plant or in animals.

The origin of mutant cells: mechanisms by which Saccharomyces cerevisiae produces cells homoplasmic for new mitochondrial mutations.

Haploid yeast cells have about 50 copies of the mitochondrial genome, and a mutational event is unlikely to affect more than one of these at a time. This raises the question of how such cells, or their progeny, become fixed (homoplasmic) for the mutant alele. We have tested the roles of six hypothetical mechanisms in producing erythromycin-resistant mutant cells: (i) random partitioning of mitochondrial genomes at cell division; (ii) intracellular selection for mtDNA molecules of one genotype; (iii) intracellular random drift of mitochondrial allele frequencies; (iv) intercellular selection for cells of a particular mitochondrial genotype; (v) induction of mitochondrial gene mutations by the antibiotic used to select mutants; and (vi) reduction in the number of mitochondrial genomes per cell by the antibiotic. Our experiments indicate that intracellular selection plays the major role in producing erythromycin-resistant mutant cells in the presence of the antibiotic. In the absence of the antibiotic, the combined effects of random drift and random partitioning are most important in determining the fate of new mutations, most of which are lost rather than fixed. Our experiments provide no evidence for mutation induction or ploidy reduction by erythromycin.

Stochastic partitioning of chloroplasts at cell division in the alga Olisthodiscus, and compensating control of chloroplast replication.

We asked how chloroplasts in a unicellular marine alga are replicated and partitioned at cell division so that each daughter cell will receive the appropriate number of copies. The data were obtained simply by counting chloroplasts in pairs of daughter cells immediately after cell division. The results show that chloroplast partitioning is not always equal; however, it is equal much more often than predicted by the binomial distribution of chloroplast numbers that would be expected if partitioning were strictly random. The parental chloroplasts were partitioned equally in approximately 76% of the divisions, while in the remaining 24% the deviations from equality were very small. To maintain a reasonable range of chloroplast numbers in the face of unequal partitioning, there must be some form of compensating control of chloroplast replication. Our data suggest that daughter cells that receive very large numbers of chloroplasts go directly to the next division without replicating their chloroplasts, while cells with very small numbers of chloroplasts go through two rounds of chloroplast replication before dividing.

Random partitioning of cytoplasmic organelles at cell division: the effect of organelle and cell volume.

When a cell divides, some cytoplasmic organelles may be partitioned randomly between the daughters. The number of organelles in each daughter is usually calculated from the binomial distribution, which assumes that the organelles occupy zero volume. We developed equations to predict numerical partitioning taking the volume of the organelles and of the cell into account. The effect of large organelle volume is that daughter cells receive equal or nearly equal numbers of organelles more often than predicted by the binomial distribution. However, numerical solutions show that volume effects are very small unless the number of organelles is very small or they occupy more than about 50% of the available cell volume.

Homoplasmic yeast cells contain no selectable "hidden" mitochondrial alleles.

Zygotes of Saccharomyces cerevisiae that are heteroplasmic for mitochondrial alleles produce diploid progeny that are homoplasmic for one allele or the other, judged by the criterion that upon further subcloning they produce daughter cells of only one phenotype or the other. Here we show that when such cells are subjected to strong selection for the missing allele, it cannot be detected, so that it is probably not present in even a single copy.

An approach to population and evolutionary genetic theory for genes in mitochondria and chloroplasts, and some results.

We developed population genetic theory for organelle genes, using an infinite alleles model appropriate for molecular genetic data, and considering the effects of mutation and random drift on the frequencies of selectively neutral alleles. The effects of maternal inheritance and vegetative segregation of organelle genes are dealt with by defining new effective gene numbers, and substituting these for 2N(e) in classical theory of nuclear genes for diploid organisms. We define three different effective gene numbers. The most general is N(lambda), defined as a function of population size, number of organelle genomes per cell, and proportions of genes contributed by male and female gametes to the zygote. In many organisms, vegetative segregation of organelle genomes and intracellular random drift of organelle gene frequencies combine to produce a predominance of homoplasmic cells within individuals in the population. Then, the effective number of organelle genes is N(eo), a simple function of the numbers of males and females and of the maternal and paternal contributions to the zygote. Finally, when the paternal contribution is very small, N( eo) is closely approximated by the number of females, N( f). Then if the sex ratio is 1, the mean time to fixation or loss of new mutations is approximately two times longer for nuclear genes than for organelle genes, and gene diversity is approximately four times greater. The difference between nuclear and organelle genes disappears or is reversed in animals in which males have large harems. The differences between nuclear and organelle gene behavior caused by maternal inheritance and vegetative segregation are generally small and may be overshadowed by differences in mutation rates to neutral alleles. For monoecious organisms, the effective number of organelle genes is approximately equal to the total population size N. We also show that a population can be effectively subdivided for organelle genes at migration rates which result in panmixis for nuclear genes, especially if males migrate more than females.

The partitioning of cytoplasmic organelles at cell division.

When an organism has only one or two mitochondria or chloroplasts per cell, it is probable that their partitioning is always stringently controlled so that each daughter cell always receives half the organelles in the parent cell. When there are more copies of an organelle, the available data suggest that partitioning is stochastic but far from random, with a strong tendency toward equality. The molecular mechanisms that promote equal partitioning are not known in any case, but the great variety of organelle behavior suggests that many different mechanisms are involved in different organisms. As Wilson (1925) pointed out, the precision of partitioning of cytoplasmic organelles rarely if ever equals that of mitosis, but it is still an expression of selection for mechanisms that will ensure the hereditary continuity of the organelles. How cells compensate for unequal partitioning by controlling organelle replication is known for only one case. But when one considers that Tetrahymena and Paramecium use different methods to compensate for unequal partitioning of macronuclear DNA, it would not be surprising if organisms use a variety of different compensating replication modes for organelles as well. What is surprising is that so little attention has been paid to these problems. Nothing could be simpler than counting organelles in dividing cells, but this has been done on a large scale in only two systems. Quantitative techniques in cell biology have been developed to the point where such studies could be done even on cells that have too many organelles for direct counting. Molecular mechanisms of partitioning have scarcely been touched on. Much more has been done on the role of the cytoskeleton in determining cell shape, and some observations have been made on its role in positioning organelles in interphase cells, but these kinds of studies have not been extended to dividing cells. Some experiments and observations have been made on the role of microtubules and microfilaments in moving cytoplasmic organelles around the cell during interphase, but again nothing has been done on their possible role in partitioning organelles at cytokinesis. The major lesson of this article is how little has been done, and how much can be done. The partitioning of cytoplasmic organelles at cell division is a wide-open field for future research, and one of great importance for both genetics and cell biology.

Persistent heteroplasmic cells for mitochondrial genes in Saccharomyces cerevisiae.

Genes in mitochondria and chloroplasts segregate rapidly during vegetative reproduction. Models to explain this vegetative segregation invoke either random segregation of organelle DNA molecules, or nonrandom segregation with random recombination events. All such models are basically stochastic. To look at vegetative segregation we took heteroplasmic (HET) cells containing mitochondrial mutations at the cap1, eryl and olil loci from several crosses. HETs were repeatedly selected and subcloned. Even after three to five successive subclonings (approximately 60-100 generations) some cells remained heteroplasmic. This confirms and extends previous observations of persistent HETs by Rank and Bech-Hansen (1972) and Forster and Kleese (1975), and by Bolen et al. (1980) for chloroplast genes in Chlamydomonas.