Extensive variation in nuclear mitochondrial DNA content between the genomes of Phytophthora sojae and Phytophthora ramorum

Fragments of mitochondrial DNA (mtDNA) transferred to the nuclear genome are called nuclear mitochondrial DNAs (NUMTs). We report here a comparison of NUMT content between genomes from two species of the same genus. Analysis of the genomes of Phytophthora sojae and P. ramorum revealed large differences in the NUMT content of the two genomes: 16.27 x 10(-3) and 2.28 x 10(-3)% of each genome, respectively. Substantial differences also exist between the two species in the sizes of the NUMTs found in each genome, with ranges of 20 to 405 bp for P. sojae and 19 to 137 bp for P. ramorum. Furthermore, in P. sojae, fragments from the mitochondrial genes rns, rnl, coxl, and nad (various subunits) are found most frequently, whereas P. ramorum NUMTs most often originate from the cox3, rpsl4, nad4, and nad5 genes. The large differences in the presumptive mtDNA insertions suggest that the insertions occurred subsequent to the divergence of the two species, and this is supported by sequence comparisons among the NUMTs and the mtDNA sequences of the two species. P. sojae mtDNA sequences inserted in the nuclear genome appear to have been altered as a result of insertions, deletions, inversions, and translocations and provide insights into active mechanisms of sequence divergence in this plant pathogen. No clear examples were found of NUMTs forming functional nuclear genes or of NUMTs inserted into exons or introns of any nuclear gene.     

Population Polymorphism of Nuclear Mitochondrial DNA Insertions Reveals Widespread Diploidy Associated with Loss of Heterozygosity in Debaryomyces hansenii

Debaryomyces hansenii, a yeast that participates in the elaboration of foodstuff, displays important genetic diversity. Our recent phylogenetic classification of this species led to the subdivision of the species into three distinct clades. D. hansenii harbors the highest number of nuclear mitochondrial DNA (NUMT) insertions known so far for hemiascomycetous yeasts. Here we assessed the intraspecific variability of the NUMTs in this species by testing their presence/absence first in 28 strains, with 21 loci previously detected in the completely sequenced strain CBS 767^T, and second in a larger panel of 77 strains, with 8 most informative loci. We were able for the first time to structure populations in D. hansenii, although we observed little NUMT insertion variability within the clades. We determined the chronology of the NUMT insertions, which turned out to correlate with the previously defined taxonomy and provided additional evidence that colonization of nuclear genomes by mitochondrial DNA is a dynamic process in yeast. In combination with flow cytometry experiments, the NUMT analysis revealed the existence of both haploid and diploid strains, the latter being heterozygous and resulting from at least four crosses among strains from the various clades. As in the diploid pathogen Candida albicans, to which D. hansenii is phylogenetically related, we observed a differential loss of heterozygosity in the diploid strains, which can explain some of the large genetic diversity found in D. hansenii over the years.Debaryomyces hansenii is a ubiquist, hemiascomycetous yeast that can be found in soil, fruits, and various manufactured foodstuff in which it participates by contributing to the maturation or as a contaminant. Its ability to grow at low temperatures and in high salinity environments makes it the most common yeast in cheeses, to which it brings a number of proteolytic and lipolytic activities and aromas in the course of maturation. D. hansenii has also been implicated as an emerging pathogen, sometimes under the name of Candida famata var. famata (see reference 17). Taxonomic classification of the species related to D. hansenii has always been subject to debate. Recent analyses have reinstated D. hansenii (previously D. hansenii var. hansenii), Debaryomyces fabryi (previously D. hansenii var. fabryi), and Debaryomyces subglobosus (previously Candida famata var. flareri) (13, 25). Phylogenetic analysis using conserved spliceosomal intron sequence comparison has shown that D. hansenii is a complex of species, which comprises at least four members: D. hansenii, Debaryomyces tyrocola, D. fabryi, and Candida flareri (previously Candida famata var. flareri) (18). In addition, our study has revealed the existence of at least three populations (clades 1 to 3) in D. hansenii, with the first one containing the strain CBS 767^T, which has been entirely sequenced (8), and the last one containing Candida famata var. famata CBS 1795.Most eukaryotic nuclear genomes contain pieces of mitochondrial sequences (designated NUMT [nuclear mitochondrial DNA] for nuclear sequences of mitochondrial origin) that result from the transfer of fragments of mitochondrial DNA (mtDNA) to the chromosomes. The number and size of the NUMTs varies greatly between eukaryotic genomes (33). A recent investigation of six hemiascomycetous yeasts has shown that even within this monophyletic group, the number of NUMTs varies greatly, from 1 in Kluyveromyces thermotolerans CBS 6340^T to 145 in D. hansenii CBS 767^T (36). The mtDNA is thought to invade nuclear genomes during the repair of chromosomal DNA double-strand breaks (DSB) by nonhomologous end joining (NHEJ), as shown experimentally in the yeast Saccharomyces cerevisiae (31, 44). The colonization of nuclear genomes by mtDNA is a dynamic evolutionary process, as observed in yeast and humans (3, 32).D. hansenii harbors the highest number of NUMTs known so far for hemiascomycetous yeasts, making it of particular interest for NUMT studies. Conversely, NUMTs are potentially interesting markers to differentiate strains of this species. The 145 NUMTs of type strain CBS 767^T are distributed in 86 loci (61 single NUMTs and 25 clusters). Most clusters (23, 25) are mosaics of NUMTs formed from noncontiguous mtDNA fragments inserted in random orientation at the same chromosomal locus. In the other two clusters, the NUMTs are all in the same orientation and order, as in the mitochondrial genome. These clusters (designated “processions”) correspond to a single ancient mtDNA insertion, followed by mutational decay, leaving recognizable mtDNA segments separated by more diverged sequences (36).Few studies have attempted to evaluate the variability of NUMTs within the same species (2, 23, 32). Here, we have studied natural isolates to assess the intraspecific variability of the NUMT insertions in the nuclear genome of the yeast species D. hansenii. We were able to structure populations in this species, to determine the chronology of the NUMT insertions, and to correlate this chronology to the taxonomy of the D. hansenii complex species. Moreover, NUMT analysis revealed the existence of both haploid and diploid strains, the latter resulting from crosses between different D. hansenii clades.     

Nuclear mitochondrial pseudogenes

As has been demonstrated recently, the transfer of genetic material from mitochondria to the nucleus and its integration into the nuclear genome is a continuous and dynamic process. Fragments of mitochondrial DNA (mtDNA) are incorporated in the nuclear genome as noncoding sequences, which are called nuclear mitochondrial pseudogenes (NUMT pseudogenes or NUMT inserts). In various eukaryotes, NUMT pseudogenes are distributed through different chromosomes to form a “library” of mtDNA fragments, providing important information on genome evolution. The escape of mtDNA from mitochondria is mostly associated with mitochondrial damage and mitophagy. Fragments of mtDNA may be integrated into nuclear DNA (nDNA) during repair of double-strand breaks (DSBs), which are caused by endogenous or exogenous agents. DSB repair of nDNA with a capture of mtDNA fragments may occur via nonhomologous end joining or a similar mechanism that involves microhomologous terminal sequences. An analysis of the available data makes it possible to suppose that the NUMT pseudogene formation rate depends on the DSB rate in nDNA, the activity of the repair systems, and the number of mtDNA fragments leaving organelles and migrating into the nucleus. Such situations are likely after exposure to damaging agents, first and foremost, ionizing radiation. Not only do new NUMT pseudogenes change the genome structure in the regions of their integration, but they may also have a significant impact on the actualization of genetic information. The de novo integration of NUMT pseudogenes in the nuclear genome may play a role in various pathologies and aging. NUMT pseudogenes may cause errors in PCR-based analyses of free mtDNA as a component of total cell DNA because of their coamplification.     

Combined phylogenetic and neighbourhood analysis of the hexose transporters and glucose sensors in yeasts

The sugar porter family in yeasts encompasses a wide variety of transporters including the hexose transporters and glucose sensors. We analysed a total of 75 members from both groups in nine hemiascomycetous species, with complete and well-annotated genomes: Saccharomyces cerevisiae, Candida glabrata, Zygosaccharomyces rouxii, Kluyveromyces thermotolerans, Saccharomyces kluyverii, Kluyveromyces lactis, Eremothecium gossypii, Debaryomyces hansenii and Yarrowia lipolytica . We present a model for the evolution of the hexose transporters and glucose sensors, supported by two types of complementary evidences: phylogeny and neighbourhood analysis. Five lineages of evolution were identified and discussed according to different mechanisms of gene evolution: lineage A for HXT1, HXT3 , HXT4, HXT5 , HXT6 and HXT7 ; lineage B for HXT2 and HXT10 ; lineage C for HXT8 ; lineage D for HXT14 ; and lineage E for SNF3 and RGT2 .     

Fidelity of capture-enrichment for mtDNA genome sequencing: influence of NUMTs

Enriching target sequences in sequencing libraries via capture hybridization to bait/probes is an efficient means of leveraging the capabilities of next-generation sequencing for obtaining sequence data from target regions of interest. However, homologous sequences from non-target regions may also be enriched by such methods. Here we investigate the fidelity of capture enrichment for complete mitochondrial DNA (mtDNA) genome sequencing by analyzing sequence data for nuclear copies of mtDNA (NUMTs). Using capture-enriched sequencing data from a mitochondria-free cell line and the parental cell line, and from samples previously sequenced from long-range PCR products, we demonstrate that NUMT alleles are indeed present in capture-enriched sequence data, but at low enough levels to not influence calling the authentic mtDNA genome sequence. However, distinguishing NUMT alleles from true low-level mutations (e.g. heteroplasmy) is more challenging. We develop here a computational method to distinguish NUMT alleles from heteroplasmies, using sequence data from artificial mixtures to optimize the method.     

Mitochondrial DNA transfer to the nucleus generates extensive insertion site variation in maize

Mitochondrial DNA (mtDNA) insertions into nuclear chromosomes have been documented in a number of eukaryotes. We used fluorescence in situ hybridization (FISH) to examine the variation of mtDNA insertions in maize. Twenty overlapping cosmids, representing the 570-kb maize mitochondrial genome, were individually labeled and hybridized to root tip metaphase chromosomes from the B73 inbred line. A minimum of 15 mtDNA insertion sites on nine chromosomes were detectable using this method. One site near the centromere on chromosome arm 9L was identified by a majority of the cosmids. To examine variation in nuclear mitochondrial DNA sequences (NUMTs), a mixture of labeled cosmids was applied to chromosome spreads of ten diverse inbred lines: A188, A632, B37, B73, BMS, KYS, Mo17, Oh43, W22, and W23. The number of detectable NUMTs varied dramatically among the lines. None of the tested inbred lines other than B73 showed the strong hybridization signal on 9L, suggesting that there is a recent mtDNA insertion at this site in B73. Different sources of B73 and W23 were examined for NUMT variation within inbred lines. Differences were detectable, suggesting either that mtDNA is being incorporated or lost from the maize nuclear genome continuously. The results indicate that mtDNA insertions represent a major source of nuclear chromosomal variation.     

Distribution of nuclear mitochondrial pseudogenes in three pollinator fig wasps associated with Ficus pumila

Nuclear mitochondrial pseudogenes (NUMTs) are nuclear sequences transferred from mitochondrial genomes. Although widespread, their distribution patterns among populations or closely related species are rarely documented. We amplified and sequenced the mitochondrial cytochrome b (Cytb) gene to check for NUMTs in three fig wasp species that pollinate Ficus pumila (Wiebesia sp. 1, 2 and 3) in Southeastern China using direct and cloned sequencing. Unambiguous sequences (332) of 487 bp in length belonging to 33 haplotypes were found by direct sequencing. Their distribution was highly concordant with those of cytochrome c oxidase subunit I (COI). Obvious signs of co-amplification of NUMTs were indicated by their uneven distribution. NUMTs were observed in all individuals of 12 populations of Wiebesia sp. 3, and 13 individuals of three northern populations of Wiebesia sp. 1. Sequencing clones of potential co-amplification products confirmed that they were NUMTs. These NUMTs either clustered as NUMT clades basal to mtDNA Cytb clades (basal NUMTs), or together with Cytb haplotypes. Basal NUMTs had either stop codons or frame-shifting mutations resulting from deletion of a 106 bp fragment. In addition, no third codon or synonymous substitutions were detected within each NUMT clade. The phylogenetic tree indicated that basal NUMTs had been inserted into nuclei before divergence of the three species. No significant pairwise differences were detected in their ratios of third codon substitutions, suggesting that these NUMTs originated from one transfer event, with duplication in the nuclear genome resulting in the coexistence of the 381 bp copy. No significant substitution differences were detected between Cytb haplotypes and NUMTs that clustered with Cytb haplotypes. However, these NUMTs coexisted with Cytb haplotypes in multiple populations, suggesting that these NUMT haplotypes were recently inserted into the nuclear genome. Both basal and recently inserted NUMTs were rare events, and were absent in most populations of Wiebesia sp. 1 and 2. Further studies are needed to distinguish between mechanisms potentially generating this rarity, such as purifying selection, genetic drift or amplification failure.     

Hit or miss in phylogeographic analyses: the case of the cryptic NUMTs

Phylogeographic studies call for attention as nuclear copies of mitochondrial DNA (NUMT) may generate erroneous results. Here, we report the presence of NUMTs differing only by 1-3 bp from authentic mitochondrial haplotypes, consequently named cryptic NUMTs. In contrast to traditional NUMTs, for which reliable tools for detection are established, cryptic NUMTs question the validity of phylogeographic analyses based solely on mitochondrial DNA, like the one presented here on the European bark beetle Ips typographus. Caution is called as cryptic NUMTs might be responsible for haplotype richness found in several species, and the necessity of refined methods for NUMT detection is highlighted.     

Complete sequences of mitochondria genomes of Aedes aegypti and Culex quinquefasciatus and comparative analysis of mitochondrial DNA fragments inserted in the nuclear genomes

We present complete sequences of the mitochondrial genomes for two important mosquitoes, Aedes aegypti and Culex quinquefasciatus, that are major vectors of dengue virus and lymphatic filariasis, respectively. The A. aegypti mitochondrial genome is 16,655 bp in length and that of C. quinquefasciatus is 15,587 bp, yet both contain 13 protein coding genes, 22 transfer RNA (tRNA) genes, one 12S ribosomal RNA (rRNA) gene, one 16S rRNA gene and a control region (CR) in the same order. The difference in the genome size is due to the difference in the length of the control region. We also analyzed insertions of nuclear copies of mtDNA-like sequences (NUMTs) in a comparative manner between the two mosquitoes. The NUMT sequences occupy ~0.008% of the A. aegypti genome and ~0.001% of the C. quinquefasciatus genome. Several NUMTs were found localized in the introns of predicted protein coding genes in both genomes (32 genes in A. aegypti but only four in C. quinquefasciatus). None of these NUMT-containing genes had an ortholog between the two species or had paralogous copies within a genome that was also NUMT-containing. It was further observed that the NUMT-containing genes were relatively longer but had lower GC content compared to the NUMT-less paralogous copies. Moreover, stretches of homologies are present among the genic and non-genic NUMTs that may play important roles in genomic rearrangement of NUMTs in these genomes. Our study provides new insights on understanding the roles of nuclear mtDNA sequences in genome complexities of these mosquitoes.     

Production of volatile compounds by cheese-ripening yeasts: requirement for a methanethiol donor for S-methyl thioacetate synthesis by Kluyveromyces lactis

Five cheese-ripening yeasts (Geotrichum candidum, Saccharomyces cerevisiae, Kluyveromyces lactis, Yarrowia lipolytica and Debaryomyces hansenii) were compared with respect to their ability to generate volatile aroma compounds. K. lactis produced a variety of esters - ethylacetate (EA) being the major one - and relatively limited amounts of volatile sulphur compounds (VSCs). Conversely, G. candidum produced significant amounts of VSCs [with the thioester S-methyl thioacetate (MTA) being the most prevalent] and lower quantities of non-sulphur volatile compounds than K. lactis. We suspect that K. lactis is able to produce and/or accumulate acetyl CoA - a common precursor of MTA and EA - but that it produces limited amounts of methanethiol (MTL); both acetyl CoA and MTL are precursors for MTA synthesis. When supplemented with exogenous MTL, MTA production greatly increased in K. lactis cultures whereas it was unchanged in G. candidum cultures, suggesting that MTL is a limiting factor for MTA synthesis in K. lactis but not in G. candidum. Our results are discussed with respect to L-methionine catabolism.     

Emergence of species-specific transporters during evolution of the hemiascomycete phylum

We have traced the evolution patterns of 2480 transmembrane transporters from five complete genome sequences spanning the entire Hemiascomycete phylum: Saccharomyces cerevisiae, Candida glabrata, Kluyveromyces lactis, Debaryomyces hansenii, and Yarrowia lipolytica. The use of nonambiguous functional and phylogenetic criteria derived from the TCDB classification system has allowed the identification within the Hemiascomycete phylum of 97 small phylogenetic transporter subfamilies comprising a total of 355 transporters submitted to four distinct evolution patterns named "ubiquitous," "species specific," "phylum gains and losses," or "homoplasic." This analysis identifies the transporters that contribute to the emergence of species during the evolution of the Hemiascomycete phylum and may aid in establishing novel phylogenetic criteria for species classification.     

Continued colonization of the human genome by mitochondrial DNA

Integration of mitochondrial DNA fragments into nuclear chromosomes (giving rise to nuclear DNA sequences of mitochondrial origin, or NUMTs) is an ongoing process that shapes nuclear genomes. In yeast this process depends on double-strand-break repair. Since NUMTs lack amplification and specific integration mechanisms, they represent the prototype of exogenous insertions in the nucleus. From sequence analysis of the genome of Homo sapiens, followed by sampling humans from different ethnic backgrounds, and chimpanzees, we have identified 27 NUMTs that are specific to humans and must have colonized human chromosomes in the last 4–6 million years. Thus, we measured the fixation rate of NUMTs in the human genome. Six such NUMTs show insertion polymorphism and provide a useful set of DNA markers for human population genetics. We also found that during recent human evolution, Chromosomes 18 and Y have been more susceptible to colonization by NUMTs. Surprisingly, 23 out of 27 human-specific NUMTs are inserted in known or predicted genes, mainly in introns. Some individuals carry a NUMT insertion in a tumor-suppressor gene and in a putative angiogenesis inhibitor. Therefore in humans, but not in yeast, NUMT integrations preferentially target coding or regulatory sequences. This is indeed the case for novel insertions associated with human diseases and those driven by environmental insults. We thus propose a mutagenic phenomenon that may be responsible for a variety of genetic diseases in humans and suggest that genetic or environmental factors that increase the frequency of chromosome breaks provide the impetus for the continued colonization of the human genome by mitochondrial DNA.     

Isolation and characterization of linear deoxyribonucleic acid plasmids from Kluyveromyces lactis and the plasmid-associated killer character.

Two linear deoxyribonucleic acid plasmids, designated pGK11 and pGK12, were isolated from the yeast Kluyveromyces lactis IFO 1267. pGK11 and pGK12 had molecular weights of 5.4 X 10(6) and 8.4 X 10(6), respectively. Both plasmids possessed the same density of 1.687 g/cm3, lighter than the densities of mitochondrial (1.692 g/cm3) and nuclear (1.699 g/cm3) deoxyribonucleic acids. A restriction map of pGK11 was constructed from digestions by EcoRI, HindIII, PstI, and BamHI. pGK12 was cleaved by EcoRI into seven fragments and by BamHI into two fragments K. lactis IFO 1267 killed Saccharomyces cerevisiae sensitive and killer strains and certain strains of Saccharomyces italicus, K. lactis, Kluyveromyces thermotolerans, and K. vanudenii. All K. lactis strains lacking the pGK1 plasmids were nonkillers. A hybrid was constructed between K. lactis IFO 1267 and a nonkiller K. lactis strain lacking the plasmids and subjected to tetrad analysis after sporulation. The killer character was extrachromosomally transmitted in all tetrads in association with the pGK1 plasmids. The double-stranded ribonucleic acid killer plasmid could not be detected in any K. lactis killer strains. It is thus highly probable that the killer character is mediated by the linear deoxyribonucleic acid plasmids. A single chromosomal gene was found which was responsible for the resistance to the K. lactis killer.     

Geotrichum candidum dominates in yeast population dynamics in Livarot, a French red-smear cheese

The diversity and dynamics of yeast populations in four batches of Livarot cheese at three points of ripening were determined. Nine different species were identified by Fourier transform infrared spectroscopy and/or sequencing, and each batch had its own unique yeast community. A real-time PCR method was developed to quantify the four main yeast species: Debaryomyces hansenii, Geotrichum candidum, Kluyveromyces sp. and Yarrowia lipolytica. Culture and molecular approaches showed that G. candidum was the dominant yeast in Livarot cheese. When D. hansenii was added as a commercial strain, it codominated with G. candidum. Kluyveromyces lactis was present only at the start of ripening. Yarrowia lipolytica appeared primarily at the end of ripening. We propose a scheme for the roles and dynamics of the principal Livarot yeasts.     

Comparative genomics of hemiascomycete yeasts: genes involved in DNA replication, repair, and recombination

Among genes conserved from bacteria to mammals are those involved in replicating and repairing DNA. Following the complete sequencing of four hemiascomycetous yeast species during the course of the Genolevures 2 project, we have studied the conservation of 106 genes involved in replication, repair, and recombination in Candida glabrata, Kluyveromyces lactis, Debaryomyces hansenii, and Yarrowia lipolytica and compared them with their Saccharomyces cerevisiae orthologues. We found that proteins belonging to the replication fork and to the nucleotide excision repair pathway were-on the average-more conserved than proteins involved in the checkpoint response to DNA damage or in meiotic recombination. The meiotic recombination proteins Spo11p and Mre11p-Rad50p, involved in making meiotic double-strand breaks (DSBs), are conserved as is Mus81p, involved in resolving meiotic recombination intermediates. Interestingly, genes found in organisms in which DSB-repair is required for proper synapsis during meiosis are also found in C. glabrata, K. lactis, and D. hansenii but not in Y. lipolytica, suggesting that two modes of meiotic recombination have been selected during evolution of the hemiascomycetous yeasts. In addition, we found that SGS1 and TOP1, respectively, a DEAD/DEAH helicase and a type I topoisomerase, are duplicated in C. glabrata and that SRS2, a helicase involved in homologous recombination, is tandemly duplicated in K. lactis. Phylogenetic analyses show that the duplicated SGS1 gene evolved faster than the original gene, probably leading to a specialization of function of the duplicated copy.     

Randomly amplified polymorphic DNA (RAPD) PCR for the identification of yeasts isolated from dairy products

In the present work randomly amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) with primers M13 and RF2 was applied to the identification at species level of yeast strains isolated from cheeses. RAPD-PCR analysis of the type strains of different yeast species gave distinctive band profiles that allowed a clear differentiation of all the considered species. Forty-two of the 48 dairy associated yeasts were clearly assigned to the species Saccharomyces cerevisiae, Kluyveromyces marxianus (anamorph Candida kefyr), Kluyveromyces lactis (anamorph Candida sphaerica), Debaryomyces hansenii (anamorph Candida famata), Yarrowia lipolytica and Torulaspora delbrueckii (anamorph Candida colliculosa). The method, which is rapid and easy to perform, could be a useful tool for the identification of yeasts present in dairy products.     

Nuclear insertions of mitochondrial origin: Database updating and usefulness in cancer studies

Nuclear insertions of mitochondrial origin (NUMTs) can be useful tools in evolution and population studies. However, due to their similarity to mitochondrial DNA (mtDNA), NUMTs may also be a source of contamination in mtDNA studies. The main goal of this work is to present a database of NUMTs, based on the latest version of the human genome—GRCh37 draft. A total of 755 insertions were identified. There are 33 paralogous sequences with over 80% sequence similarity and of a greater length than 500 bp. The non-identical positions between paralogous sequences are listed for the first time. As an application example, the described database is used to evaluate the impact of NUMT contamination in cancer studies. The evaluation reveals that 220 positions from 256 with zero hits in the current mtDNA phylogeny could in fact be traced to one or more nuclear insertions of mtDNA. This is due to they are located in non-identical positions between mtDNA and nuclear DNA (nDNA). After in silico primer validation of each revised cancer study, risk of co-amplification between mtDNA and nDNA was detected in some cases, whereas in others no risk of amplification was identified. This approach to cancer studies clearly proves the potential of our NUMT database as a valuable new tool to validate mtDNA mutations described in different contexts. Moreover, due to the amount of information provided for each nuclear insertion, this database should play an important role in designing evolutionary, phylogenetic and epidemiological studies.     

NUMTs in sequenced eukaryotic genomes

Mitochondrial DNA sequences are frequently transferred to the nucleus giving rise to the so-called nuclear mitochondrial DNA (NUMT). Analysis of 13 eukaryotic species with sequenced mitochondrial and nuclear genomes reveals a large interspecific variation of NUMT number and size. Copy number ranges from none or few copies in Anopheles, Caenorhabditis, Plasmodium, Drosophila, and Fugu to more than 500 in human, rice, and Arabidopsis. The average size is between 62 (baker's yeast) and 647 bps (Neurospora), respectively. A correlation between the abundance of NUMTs and the size of the nuclear or the mitochondrial genomes, or of the nuclear gene density, is not evident. Other factors, such as the number and/or stability of mitochondria in the germline, or species-specific mechanisms controlling accumulation/loss of nuclear DNA, might be responsible for the interspecific diversity in NUMT accumulation.     

Mutations in Mgi Genes Convert Kluyveromyces Lactis into a Petite-Positive Yeast

Following targeted disruption of the unique CYC1 gene, the petite-negative yeast, Kluyveromyces lactis, was found to grow fermentatively in the absence of cytochrome c-mediated respiration. This observation encouraged us to seek mitochondrial mutants by treatment of K. lactis with ethidium bromide at the highest concentration permitting survival. By this technique, we isolated four mtDNA mutants, three lacking mtDNA and one with a deleted mitochondrial genome. In the three isolates lacking mtDNA, a nuclear mutation is present that permits petite formation. The three mutations occur at two different loci, designated MGI1 and MGI2 (for Mitochondrial Genome Integrity). The mgi mutations convert K. lactis into a petite-positive yeast. Like bakers' yeast, the mgi mutants spontaneously produce petites with deletions in mtDNA and lose this genome at high frequency on treatment with ethidium bromide. We suggest that the MGI gene products are required for maintaining the integrity of the mitochondrial genome and that, petite-positive yeasts may be naturally altered in one or other of these genes.     

The composition of Camembert cheese-ripening cultures modulates both mycelial growth and appearance

The fungal microbiota of bloomy-rind cheeses, such as Camembert, forms a complex ecosystem that has not been well studied, and its monitoring during the ripening period remains a challenge. One limitation of enumerating yeasts and molds on traditional agar media is that hyphae are multicellular structures, and colonies on a petri dish rarely develop from single cells. In addition, fungi tend to rapidly invade agar surfaces, covering small yeast colonies and resulting in an underestimation of their number. In this study, we developed a real-time quantitative PCR (qPCR) method using TaqMan probes to quantify a mixed fungal community containing the most common dairy yeasts and molds: Penicillium camemberti, Geotrichum candidum, Debaryomyces hansenii, and Kluyveromyces lactis on soft-cheese model curds (SCMC). The qPCR method was optimized and validated on pure cultures and used to evaluate the growth dynamics of a ripening culture containing P. camemberti, G. candidum, and K. lactis on the surface of the SCMC during a 31-day ripening period. The results showed that P. camemberti and G. candidum quickly dominated the ecosystem, while K. lactis remained less abundant. When added to this ecosystem, D. hansenii completely inhibited the growth of K. lactis in addition to reducing the growth of the other fungi. This result was confirmed by the decrease in the mycelium biomass on SCMC. This study compares culture-dependent and qPCR methods to successfully quantify complex fungal microbiota on a model curd simulating Camembert-type cheese.