Replication and preferential inheritance of hypersuppressive petite mitochondrial DNA

Wild-type yeast mitochondrial DNA (mtDNA) is inherited biparentally, whereas mtDNA of hypersuppressive petite mutants is inherited uniparentally in crosses to strains with wild-type mtDNA. Genomes of hypersuppressive petites contain a conserved ori sequence that includes a promoter, but it is unclear whether the ori confers a segregation or replication advantage. Fluorescent in situ hybridization analysis of wild-type and petite mtDNAs in crosses reveals no preferential segregation of hypersuppressive petite mtDNA to first zygotic buds. We identify single-stranded DNA circles and RNA-primed DNA replication intermediates in hypersuppressive petite mtDNA that are absent from non-hypersuppressive petites. Mutating the promoter blocks hypersuppressiveness in crosses to wild-type strains and eliminates the distinctive replication intermediates. We propose that promoter-dependent RNA-primed replication accounts for the uniparental inheritance of hypersuppressive petite mtDNA.     

The mitochondrial nucleoid protein, Mgm101p, of Saccharomyces cerevisiae is involved in the maintenance of rho(+) and ori/rep-devoid petite genomes but is not required for hypersuppressive rho(-) mtDNA

The Saccharomyces cerevisiae MGM101 gene encodes a DNA-binding protein targeted to mitochondrial nucleoids. MGM101 is essential for maintenance of a functional rho(+) genome because meiotic segregants, with a disrupted mgm101 allele, cannot undergo more than 10 divisions on glycerol medium. Quantitative analysis of mtDNA copy number in a rho(+) strain carrying a temperature-sensitive allele, mgm101-1, revealed that the amount of mtDNA is halved each cell division upon a shift to the restrictive temperature. These data suggest that mtDNA replication is rapidly blocked in cells lacking MGM101. However, a small proportion of meiotic segregants, disrupted in MGM101, have rho(-) genomes that are stably maintained. Interestingly, all surviving rho(-) mtDNAs contain an ori/rep sequence. Disruption of MGM101 in hypersuppressive (HS) strains does not have a significant effect on the propagation of HS rho(-) mtDNA. However, in petites lacking an ori/rep, disruption of MGM101 leads to either a complete loss or a dramatically decreased stability of mtDNA. This discriminatory effect of MGM101 suggests that replication of rho(+) and ori/rep-devoid rho(-) mtDNAs is carried out by the same process. By contrast, the persistence of ori/rep-containing mtDNA in HS petites lacking MGM101 identifies a distinct replication pathway. The alternative mtDNA replication mechanism provided by ori/rep is independent of mitochondrial RNA polymerase encoded by RPO41 as a HS rho(-) genome is stably maintained in a mgm101, rpo41 double mutant.     

The transmission disadvantage of yeast mitochondrial intergenic mutants is eliminated in the mgt1 (cce1) background.

A trans-acting element, MGT1 (also called CCE1), has previously been shown to be required in Saccharomyces cerevisiae for the preferential transmission of petite mitochondrial DNA (mtDNA) molecules over wild-type mtDNA molecules. In the present study a possible role of this nuclear gene in the transmission of mtDNA from various respiration-competent mutants was studied. Several of these mutants, lacking one or the other of two biologically active mitochondrial intergenic sequences, were employed in genetic crosses. When these deletion mutants were crossed to the parental wild-type strain in the MGT1/CCE1 background, the progeny contained predominantly wild-type mtDNA molecules. When crosses were performed in the mgt1/cce1 background, the parental molecules interacted in zygotes and underwent homologous recombination but wild-type and intergenic-deletion alleles were transmitted with equal frequencies.     

Cloning of neuronal mtDNA variants in cultured cells by synaptosome fusion with mtDNA-less cells

Synaptosome cybrids were used to confirm the presence of heteroplasmic mtDNA sequence variants in the human brain. Synaptosomes contain one to several mitochondria, and when fused to mtDNA-deficient (ρ°) mouse or human cell lines result in viable cybrid cell lines. The brain origin of mouse synaptosome cybrid mtDNAs was confirmed using sequence polymorphisms in the mtDNA COIII, ND3 and tRNA^Arg genes. The brain origin of the human synaptosome cybrids was confirmed using a rare mtDNA MboI polymorphism. Fusion of synaptosomes from the brain of a 35-year-old woman resulted in 71 synaptosome cybrids. Sequencing the mtDNA control region of these cybrid clones revealed differences in the number of Cs in a poly C track between nucleotide pairs (nps) 301 and 309. Three percent of the cybrid clones had mtDNAs with 10 Cs, 76% had nine, 18% had eight and 3% had seven Cs. Comparable results were obtained by PCR amplification, cloning and sequencing of mtDNA control regions directly from the patient’s brain tissue, but not when the control region was amplified and cloned from a synaptosome cybrid homoplasmic for a mtDNA with nine Cs. Thus, we have clonally recovered mtDNA control region length variants from an adult human brain without recourse to PCR, and established the variant mtDNAs within living cultured cells. This confirms that some mtDNA heteroplasmy can exist in human neurons, and provides the opportunity to study its functional significance.     

Genetic exchange and recombination in populations of the root-infecting fungus Armillaria gallica

Genetic individuals, or genets, of Armillaria and other root-infecting basidiomycetes are usually found in discrete patches that often include the root systems of several adjacent trees. Each diploid individual is thought to arise in an unique mating event and then grow vegetatively in an expanding territory over a long period of time. Our objective in this study was to describe the population from which such genetic individuals are drawn. In a sample including 274 collections representing 121 genetic individuals of A. gallica (synonym A. bulbosa ) from two sites in each of four regions of eastern North America, genotype frequencies at seven nuclear loci were not significantly different from Hardy-Weinberg expectations. Furthermore, allele frequencies at the seven loci were not significantly different between regions. Additional allelic data from four non-contiguous regions of mitochondrial DNA showed little or no population subdivision over the four regions. Analysis of the distribution of multilocus mtDNA haplotypes revealed some clonal transmission of mtDNAs between genets and nonrandom mating within sites. Despite the sharing of mtDNA types by some individuals, the overall sample contained a high level of genotypic diversity. The apparent linkage equilibrium between some pairs of loci and the high level of phylogenetic inconsistency among all four loci suggest the occurrence heteroplasmy and recombination among mtDNAs of A. gallica in nature. In laboratory matings of two haploid strains with different mtDNA types, a low frequency of recombination in mtDNA was detected.     

Din7 and Mhr1 expression levels regulate double-strand-break–induced replication and recombination of mtDNA at ori5 in yeast

The Ntg1 and Mhr1 proteins initiate rolling-circle mitochondrial (mt) DNA replication to achieve homoplasmy, and they also induce homologous recombination to maintain mitochondrial genome integrity. Although replication and recombination profoundly influence mitochondrial inheritance, the regulatory mechanisms that determine the choice between these pathways remain unknown. In Saccharomyces cerevisiae, double-strand breaks (DSBs) introduced by Ntg1 at the mitochondrial replication origin ori5 induce homologous DNA pairing by Mhr1, and reactive oxygen species (ROS) enhance production of DSBs. Here, we show that a mitochondrial nuclease encoded by the nuclear gene DIN7 (DNA damage inducible gene) has 5′-exodeoxyribonuclease activity. Using a small ρ⁻ mtDNA bearing ori5 (hypersuppressive; HS) as a model mtDNA, we revealed that DIN7 is required for ROS-enhanced mtDNA replication and recombination that are both induced at ori5. Din7 overproduction enhanced Mhr1-dependent mtDNA replication and increased the number of residual DSBs at ori5 in HS-ρ⁻ cells and increased deletion mutagenesis at the ori5 region in ρ⁺ cells. However, simultaneous overproduction of Mhr1 suppressed all of these phenotypes and enhanced homologous recombination. Our results suggest that after homologous pairing, the relative activity levels of Din7 and Mhr1 modulate the preference for replication versus homologous recombination to repair DSBs at ori5.     

Evolutionarily stable mutation rate in a periodically changing environment

Evolution of mutation rate controlled by a neutral modifier is studied for a locus with two alleles under temporally fluctuating selection pressure. A general formula is derived to calculate the evolutionarily stable mutation rate μ(ess) in an infinitely large haploid population, and following results are obtained. (I) For any fluctuation, periodic or random: (1) if the recombination rate r per generation between the modifier and the main locus is 0, μ(ess) is the same as the optimal mutation rate μ(op) which maximizes the long-term geometric average of population fitness; and (2) for any r, if the strength s of selection per generation is very large, μ(ess) is equal to the reciprocal of the average number T of generations (duration time) during which one allele is persistently favored than the other. (II) For a periodic fluctuation in the limit of small s and r, μ(ess)T is a function of sT and rT with properties: (1) for a given sT, μ(ess)T decreases with increasing rT; (2) for sT </= 1, μ(ess)T is almost independent of sT, and depends on rT as μ(ess)T & 1.6 for rT << 1 and μ(ess)T & 6/rT for rT >> 1; and (3) for sT >/= 1, and for a given rT, μ(ess)T decreases with increasing sT to a certain minimum less than 1, and then increases to 1 asymptotically in the limit of large sT. (III) For a fluctuation consisting of multiple Fourier components (i.e., sine wave components), the component with the longest period is the most effective in determining μ(ess) (low pass filter effect). (IV) When the cost c of preventing mutation is positive, the modifier is nonneutral, and μ(ess) becomes larger than in the case of neutral modifier under the same selection pressure acting at the main locus. The value of c which makes μ(ess) equal to μ(op) of the neutral modifier case is calculated. It is argued that this value gives a critical cost such that, so long as the actual cost exceeds this value, the evolution rate at the main locus must be smaller than its mutation rate μ(ess).     

Reactive oxygen species regulate DNA copy number in isolated yeast mitochondria by triggering recombination-mediated replication

Mitochondrial DNA (mtDNA) encodes proteins that are essential for cellular ATP production. Reactive oxygen species (ROS) are respiratory byproducts that damage mtDNA and other cellular components. In Saccharomyces cerevisiae, the oxidized base excision-repair enzyme Ntg1 introduces a double-stranded break (DSB) at the mtDNA replication origin ori5; this DSB initiates the rolling-circle mtDNA replication mediated by the homologous DNA pairing protein Mhr1. Thus, ROS may play a role in the regulation of mtDNA copy number. Here, we show that the treatment of isolated mitochondria with low concentrations of hydrogen peroxide increased mtDNA copy number in an Ntg1- and Mhr1-dependent manner. This treatment elevated the DSB levels at ori5 of hypersuppressive [rho^–] mtDNA only if Ntg1 was active. In vitro Ntg1-treatment of hypersuppressive [rho^–] mtDNA extracted from hydrogen peroxide-treated mitochondria revealed increased oxidative modifications at ori5 loci. We also observed that purified Ntg1 created breaks in single-stranded DNA harboring oxidized bases, and that ori5 loci have single-stranded character. Furthermore, chronic low levels of hydrogen peroxide increased in vivo mtDNA copy number. We therefore propose that ROS act as a regulator of mtDNA copy number, acting through the Mhr1-dependent initiation of rolling-circle replication promoted by Ntg1-induced DSB in the single-stranded regions at ori5.     

Mode of Action Studies on a Chiral Diphenyl Ether Peroxidizing Herbicide: Correlation between Differential Inhibition of Protoporphyrinogen IX Oxidase Activity and Induction of Tetrapyrrole Accumulation by the Enantiomers

The nitrodiphenyl ether herbicide 5-[2-chloro-4-(trifluoromethyl)phenoxy]-2-nitroacetophenone oxime-O-(acetic acid, methyl ester) (DPEI) induced an abnormal accumulation of protoporphyrin IX in darkness in the green alga Chlamydomonas reinhardtii, as determined by high-performance liquid chromatography and spectrofluorimetry. It also inhibited the increase in cell density of the alga in light-grown cultures with an I₅₀ (concentration required to decrease cell density increase to 50% of the noninhibited control value) of 0.16 μm. The relative ability of four peroxidizing diphenyl ether herbicides to cause tetrapyrrole accumulation in C. reinhardtii correlated qualitatively with their ability to inhibit the increase in cell density in light-grown cultures. The purified S(−) enantiomer of the optically active phthalide DPE 5-[2-chloro-4-(trifluoromethyl)phenoxy]-3-methylphthalide (DPEIII), which has greater herbicidal activity than the R(+) isomer, induces a 4- to 5-fold greater tetrapyrrole accumulation than the R(+) isomer. The I₅₀ for inhibition of increase in cell density in light-grown cultures of C. reinhardtii by the S(−) isomer (0.019 μm) is less than 25% of that for the R(+) isomer. DPEIII inhibits protoporphyrinogen IX oxidase activity in pea (Pisum sativum) etioplast lysates, with the S(−) enantiomer showing considerably greater potency than the R(+) isomer and the racemic mixture showing a potency intermediate between the two. The results indicate that the site at which DPEs inhibit protoporphyrinogen IX oxidase shows chiral discrimination and provide further evidence for the link between inhibition of this enzyme, protoporphyrin IX accumulation, and the phytotoxicity of DPE herbicides.     

The Spread of an Advantageous Allele across a Barrier: The Effects of Random Drift and Selection against Heterozygotes

A local barrier to gene flow will delay the spread of an advantageous allele. Exact calculations for the deterministic case show that an allele that is favorable when rare is delayed very little even by a strong barrier: its spread is slowed by a time proportional to log ((B/σ) &2S)/S, where B is the barrier strength, σ the dispersal range, and fitnesses are 1:1 + S:1 + 2S. However, when there is selection against heterozygotes, such that the allele cannot increase from low frequency, a barrier can cause a much greater delay. If gene flow is reduced below a critical value, spread is entirely prevented. Stochastic simulations show that with additive selection, random drift slows down the spread of the allele, below the deterministic speed of σ &2S. The delay to the advance of an advantageous allele caused by a strong barrier can be substantially increased by random drift and increases with B/(2Sρσ(2)) in a one-dimensional habitat of density ρ. However, with selection against heterozygotes, drift can facilitate the spread and can free an allele that would otherwise be trapped indefinitely by a strong barrier. We discuss the implications of these results for the evolution of chromosome rearrangements.     

DNA base excision repair activities and pathway function in mitochondrial and cellular lysates from cells lacking mitochondrial DNA

Mitochondrial DNA (mtDNA) contains higher steady-state levels of oxidative damage and mutates at rates significantly greater than nuclear DNA. Oxidative lesions in mtDNA are removed by a base excision repair (BER) pathway. All mtDNA repair proteins are nuclear encoded and imported. Most mtDNA repair proteins so far discovered are either identical to nuclear DNA repair proteins or isoforms of nuclear proteins arising from differential splicing. Regulation of mitochondrial BER is therefore not expected to be independent of nuclear BER, though the extent to which mitochondrial BER is regulated with respect to mtDNA amount or damage is largely unknown. Here we have measured DNA BER activities in lysates of mitochondria isolated from human 143B TK^– osteosarcoma cells that had been depleted of mtDNA (ρ⁰) or not (wt). Despite the total absence of mtDNA in the ρ⁰ cells, a complete mitochondrial BER pathway was present, as demonstrated using an in vitro assay with synthetic oligonucleotides. Measurement of individual BER protein activities in mitochondrial lysates indicated that some BER activities are insensitive to the lack of mtDNA. Uracil and 8-oxoguanine DNA glycosylase activities were relatively insensitive to the absence of mtDNA, only about 25% reduced in ρ⁰ relative to wt cells. Apurinic/apyrimidinic (AP) endonuclease and polymerase γ activities were more affected, 65 and 45% lower, respectively, in ρ⁰ mitochondria. Overall BER activity in lysates was also about 65% reduced in ρ⁰ mitochondria. To identify the limiting deficiencies in BER of ρ⁰ mitochondria we supplemented the BER assay of mitochondrial lysates with pure uracil DNA glycosylase, AP endonuclease and/or the catalytic subunit of polymerase γ. BER activity was stimulated by addition of uracil DNA glycosylase and polymerase γ. However, no addition or combination of additions stimulated BER activity to wt levels. This suggests that an unknown activity, factor or interaction important in BER is deficient in ρ⁰ mitochondria. While nuclear BER protein levels and activities were generally not altered in ρ⁰ cells, AP endonuclease activity was substantially reduced in nuclear and in whole cell extracts. This appeared to be due to reduced endogenous reactive oxygen species (ROS) production in ρ⁰ cells, and not a general dysfunction of ρ⁰ cells, as exposure of cells to ROS rapidly stimulated increases in AP endonuclease activities and APE1 protein levels.     

Leishmania major CorA-like magnesium transporters play a critical role in parasite development and virulence

Establishment of infection by Leishmania depends on the transformation of the invading metacyclic promastigotes into the obligatory intracellular amastigotes, and their subsequent survival in the macrophage phagolysosome, which is low in magnesium. We show that two Leishmania major proteins designated MGT1 and MGT2, which play a critical role in these processes, belong to the two-transmembrane domain (2-TM-GxN) cation transporter family and share homology with the major bacterial magnesium transporter CorA. Although both are present in the endoplasmic reticulum throughout the life cycle of the parasite, MGT1 is more highly expressed in the infectious metacyclic parasites, while MGT2 is enriched in the immature procyclic stages. The two proteins, although predicted to be structurally similar, have features that suggest different regulatory or gating mechanisms. The two proteins may also be functionally distinct, since only MGT1 complements an Escherichia coliΔCorA mutant. In addition, deletion of one mgt1 allele from L. major led to increased virulence, while deletion of one allele of mgt2 resulted in slower growth and total loss of virulence in vitro and in vivo. This loss of virulence may be due to an impaired transformation of the parasites into amastigotes. Deletion of both mgt1 alleles in the hemizygous MGT2 knockdown parasites reversed the growth defect and partially restored virulence. Our data indicate that the MGTs play a critical role in parasite growth, development and virulence.     

Mitochondrial DNA Genetics and the Heteroplasmy Conundrum in Evolution and Disease

The unorthodox genetics of the mtDNA is providing new perspectives on the etiology of the common “complex” diseases. The maternally inherited mtDNA codes for essential energy genes, is present in thousands of copies per cell, and has a very high mutation rate. New mtDNA mutations arise among thousands of other mtDNAs. The mechanisms by which these “heteroplasmic” mtDNA mutations come to predominate in the female germline and somatic tissues is poorly understood, but essential for understanding the clinical variability of a range of diseases. Maternal inheritance and heteroplasmy also pose major challengers for the diagnosis and prevention of mtDNA disease.     

Somatic mtDNA Mutation Spectra in the Aging Human Putamen

The accumulation of heteroplasmic mitochondrial DNA (mtDNA) deletions and single nucleotide variants (SNVs) is a well-accepted facet of the biology of aging, yet comprehensive mutation spectra have not been described. To address this, we have used next generation sequencing of mtDNA-enriched libraries (Mito-Seq) to investigate mtDNA mutation spectra of putamen from young and aged donors. Frequencies of the “common” deletion and other “major arc” deletions were significantly increased in the aged cohort with the fold increase in the frequency of the common deletion exceeding that of major arc deletions. SNVs also increased with age with the highest rate of accumulation in the non-coding control region which contains elements necessary for translation and replication. Examination of predicted amino acid changes revealed a skew towards pathogenic SNVs in the coding region driven by mutation bias. Levels of the pathogenic m.3243A>G tRNA mutation were also found to increase with age. Novel multimeric tandem duplications that resemble murine control region multimers and yeast ρ⁻ mtDNAs, were identified in both young and aged specimens. Clonal ∼50 bp deletions in the control region were found at high frequencies in aged specimens. Our results reveal the complex manner in which the mitochondrial genome alters with age and provides a foundation for studies of other tissues and disease states.     

Mitochondrial DNA reveals formation of nonhybrid frogs by natural matings between hemiclonal hybrids.

The European water frog Rana esculenta (RL), a natural hybrid between R. ridibunda (RR) and R. lessonae (LL), reproduces by hybridogenesis: haploid gametes usually contain an intact chromosome set of R. ridibunda (R); the lessonae nuclear genome (L) is lost from the germ line. Hybridity is restored in the next generation, via fertilization by syntopic R. lessonae. Matings between two hybrids (RL x RL) usually give inviable R. ridibunda (RR) progeny. The adult R. ridibunda subpopulation of Trubeschloo, a gravel pit in northern Switzerland, consists only of females. Fragment patterns for mitochondrial DNA (mtDNA) of these R. ridibunda were identical with those of syntopic R. esculenta and of local populations of R. lessonae; they differed from the patterns in eastern European populations of R. lessonae and of R. ridibunda mtDNAs (3.7% and 9.3% estimated sequence divergence, respectively). In contrast, mtDNAs of two R. ridibunda from an introduced Swiss population with both sexes, although different (2.7% divergence) from each other, were typical R. ridibunda rather than R. lessonae mtDNAs. These data, together with unisexuality, demonstrate conclusively that the all-female R. ridibunda population at Trubeschloo originated from matings between two R. esculenta. The formation of independently reproducing R. ridibunda populations via such hybrid x hybrid matings is precluded because progeny of these matings are unisexual. Recombination in the regenerated fertile R. ridibunda females, followed by matings with R. lessonae, nevertheless provides a mechanism for meiotic reshuffling of genetic material in ridibunda haplotypes that is not typically available in hemiclonal lineages.     

Yeast Cells Expressing the Human Mitochondrial DNA Polymerase Reveal Correlations between Polymerase Fidelity and Human Disease Progression

Mutations in the human mitochondrial polymerase (polymerase-γ (Pol-γ)) are associated with various mitochondrial disorders, including mitochondrial DNA (mtDNA) depletion syndrome, Alpers syndrome, and progressive external opthamalplegia. To correlate biochemically quantifiable defects resulting from point mutations in Pol-γ with their physiological consequences, we created “humanized” yeast, replacing the yeast mtDNA polymerase (MIP1) with human Pol-γ. Despite differences in the replication and repair mechanism, we show that the human polymerase efficiently complements the yeast mip1 knockouts, suggesting common fundamental mechanisms of replication and conserved interactions between the human polymerase and other components of the replisome. We also examined the effects of four disease-related point mutations (S305R, H932Y, Y951N, and Y955C) and an exonuclease-deficient mutant (D198A/E200A). In haploid cells, each mutant results in rapid mtDNA depletion, increased mutation frequency, and mitochondrial dysfunction. Mutation frequencies measured in vivo equal those measured with purified enzyme in vitro. In heterozygous diploid cells, wild-type Pol-γ suppresses mutation-associated growth defects, but continuous growth eventually leads to aerobic respiration defects, reduced mtDNA content, and depolarized mitochondrial membranes. The severity of the Pol-γ mutant phenotype in heterozygous diploid humanized yeast correlates with the approximate age of disease onset and the severity of symptoms observed in humans.     

Evaluation of Group Genetic Ancestry of Populations from Philadelphia and Dakar in the Context of Sex-Biased Admixture in the Americas

Background

Population history can be reflected in group genetic ancestry, where genomic variation captured by the mitochondrial DNA (mtDNA) and non-recombining portion of the Y chromosome (NRY) can separate female- and male-specific admixture processes. Genetic ancestry may influence genetic association studies due to differences in individual admixture within recently admixed populations like African Americans.

Principal Findings

We evaluated the genetic ancestry of Senegalese as well as European Americans and African Americans from Philadelphia. Senegalese mtDNA consisted of ∼12% U haplotypes (U6 and U5b1b haplotypes, common in North Africa) while the NRY haplotypes belonged solely to haplogroup E. In Philadelphia, we observed varying degrees of admixture. While African Americans have 9–10% mtDNAs and ∼31% NRYs of European origin, these results are not mirrored in the mtDNA/NRY pools of European Americans: they have less than 7% mtDNAs and less than 2% NRYs from non-European sources. Additionally, there is <2% Native American contribution to Philadelphian African American ancestry and the admixture from combined mtDNA/NRY estimates is consistent with the admixture derived from autosomal genetic data. To further dissect these estimates, we have analyzed our samples in the context of different demographic groups in the Americas.

Conclusions

We found that sex-biased admixture in African-derived populations is present throughout the Americas, with continual influence of European males, while Native American females contribute mainly to populations of the Caribbean and South America. The high non-European female contribution to the pool of European-derived populations is consistently characteristic of Iberian colonization. These data suggest that genomic data correlate well with historical records of colonization in the Americas.     

How Often Do Duplicated Genes Evolve New Functions?

A recently duplicated gene can either fix a null allele (becoming a pseudogene) or fix an (advantageous) allele giving a slightly different function, starting it on the road to evolving a new function. Here we examine the relative probabilities of these two events under a simple model. Null alleles are assumed to be neutral; linkage effects are ignored, as are unequal crossing over and gene conversion. These assumptions likely make our results underestimates for the probability that an advantageous allele is fixed first. When new advantageous mutations are additive with selection coefficient s and the ratio of advantageous to null mutations is ρ, the probability an advantageous allele is fixed first is ([1 - e(-S)]/[ρS] + 1)(-1), where S = 4N(e)s with N(e) the effective population size. The probability that a duplicate locus becomes a pseudogene, as opposed to evolving a new gene function, is high unless ρS & 1. However, even if advantageous mutations are very rare relative to null mutations, for sufficiently large populations ρS & 1 and new gene function, rather than pseudogene formation, is the expected fate of most duplicated genes.     

Evidence of the Insensitivity of the alpha-inc Allele to the Function of the Homothallic Genes in Saccharomyces Yeasts

The possible function of the α-inc allele (an α mating-type allele that is insensitive to the function of the homothallic gene system) was investigated by means of protoplast fusion. The fusion of protoplasts prepared from haploid strains of α-inc HO HMα HMa and α ho hmα HMa gave rise mainly to nonmating clones (58 of 64 isolates) and a few clones (six of 64 isolates) showing α mating type. Thirty of the 58 nonmating clones showed the diploid cell size and 28 clones had a larger cell size. Tetrad analysis of the nonmating clones with diploid cell size indicated that they were a/α-inc diploid; the normal α allele in α/α-inc cells was preferentially switched to an a allele. This observation further indicated that the HO/ho HMα/hmα HMa/HMa genotype is effective for the conversion of the α to a and that the inconvertibility of the α-inc allele is due to the insensitivity of the mating-type allele to the functional combination of the homothallic genes. It was suspected that fusion products larger than diploid cells might have been caused by multiple fusion of protoplasts.