Phylogeny of the Large Extrachromosomal DNA of Organisms in the Phylum Apicomplexa

ABSTRACT. Organisms in the phylum Apicomplexa appear to have a large extrachromosomal DNA which is unrelated to the mitochondrial DNA. Based on the apparent gene content of the large (35 kb) extrachromosomal DNA of Plasmodium falciparum , it has been suggested that it is a plastid-like DNA, which may be related to the plastid DNA of rhodophytes. However, phylogenetic analyses have been inconclusive. It has been suggested that this is due to the unusually high A + T content of the Plasmodium falciparum large extrachromosomal DNA. To further investigate the evolution of the apicomplexan large extrachromosomal DNA, the DNA sequence of the organellar ribosomal RNA gene from Toxoplasma gondii , was determined. The Toxoplasma gondii rDNA sequence was most similar to the large extrachromosomal rDNA of Plasmodium falciparum , but was much less A + T rich. Phylogenetic analyses were carried out using the LogDet transformation to minimize the impact of nucleotide bias. These studies support the evolutionary relatedness of the Toxoplasma gondii rDNA with the large extrachromosomal rDNA of Plasmodium falciparum and with the organellar rDNA of another parasite in the phylum Apicomplexa, Babesia bovis. These analyses also suggest that the apicomplexan large extra-chromosomal DNA may be more closely related to the plastid DNA of euglenoids than to those of rhodophytes.     

Plastid and nuclear DNA synthesis are not coupled in suspension cells ofNicotiana tabacum

The relationship between nuclear and plastid DNA synthesis in cultured tobacco cells was measured by following³H-thymidine incorporation into total cellular DNA in the absence or presence of specific inhibitors. Plastid DNA synthesis was determined by hybridization of total radiolabeled cellular DNA to cloned chloroplast DNA. Cycloheximide, an inhibitor of nuclear encoded cytoplasmic protein synthesis, caused a rapid and severe inhibition of nuclear DNA synthesis and a delayed inhibition of plastid DNA synthesis. By contrast, chloramphenicol which only inhibits plastid and mitochondrial protein production, shows little inhibition of either nuclear or plastid DNA synthesis even after 24 h of exposure to the cells. The inhibition of nuclear DNA synthesis by aphidicolin, which specifically blocks the nuclear DNA polymeraseα, has no significant effect on plastid DNA formation. Conversely, the restraint of plastid DNA synthesis exerted by low levels of ethidium bromide has no effect on nuclear DNA synthesis. These results show that the synthesis of plastid and nuclear DNA are not coupled to one another. However, both genomes require the formation of cytoplasmic proteins for their replication, though our data suggest that different proteins regulate the biosynthesis of nuclear and plastid DNA.     

Organelle DNAs: The bit players in malaria parasite DNA replication

The replication mechanics of the extrachromosomal DNAs of the malaria parasite are beginning to be anravelled. At 6 kb, the mitochondrial genome is the smallest known and, unlike higher eukaryotes, its multiple copies per cell occur as polydisperse linear concatemers. Here, Don Williamson, Peter Preiser and Iain Wilson discuss recent evidence that this DNA replicates by a process akin to those of certain bacteriophages, which make use of extensive recombination coupled with rolling circles. The parasite's second extrachromosomal DNA, a 35 kb circular molecule thought to be a plastid remnant inherited from a remote photoautotroph, probably replicates in a more familiar fashion from conventional origins or D loops. Improved understanding of both organelle's replicative mechanisms could give new leads to malaria chemotherapy.     

Chloroplast DNA synthesis during the cell cycle in cultured cells of Nicotiana tabacum: inhibition by nalidixic acid and hydroxyurea

The effects of nalidixic acid and hydroxyurea on nuclear and chloroplast DNA formation in cultured cells of Nicotiana tabacum were investigated. At low concentrations (5 and 20 micrograms/ml) nalidixic acid, an inhibitor of DNA gyrase, exhibited a greater inhibitory effect on plastid DNA synthesis than on nuclear DNA formation. Since the plastid genome is a circular double-stranded DNA, this is consistent with the proven involvement of a DNA gyrase in the replication of closed circular duplex DNA genomes in procaryotic cells. At a high concentration of nalidixic acid (50 micrograms/ml), DNA synthesis in both the plastid and nuclear compartment was rapidly inhibited. Removal of the drug from the culture medium led to the resumption of DNA synthesis in 8 h. Hydroxyurea, an inhibitor of ribonucleoside diphosphate reductase, also depresses nuclear as well as plastid DNA formation. Removal of hydroxyurea from the blocked cells leads to a burst of nuclear DNA synthesis, suggesting that the cells had been synchronized at the G1/S boundary. The recovery of plastid DNA synthesis occurs within the same time frame as that of nuclear DNA. However, whereas plastid DNA formation is then maintained at a constant rate, nuclear DNA synthesis reaches a peak and subsequently declines. These results indicate that the synthesis of plastid DNA is independent of the cell cycle events governing nuclear DNA formation in cultured plant cells.     

Plastid origin of an extrachromosomal DNA molecule from Plasmodium, the causative agent of malaria.

Several species of Plasmodium have been shown to contain a circular extrachromosomal DNA molecule which is widely supposed to be mitochondrial DNA. However, it has recently been shown to have a number of features in common with chloroplast DNA. Here, a phylogenetic analysis of RNA polymerase coding sequences from the Plasmodium molecule has been carried out using distance matrix, maximum likelihood, parsimony and operator invariant methods. The analysis indicates that the molecule is in fact derived from an oxygenic photosynthetic organism and should be regarded as plastid DNA. This suggests that Plasmodium originated from a phototroph that has lost the capacity to photosynthesize.     

Evidence against the involvement of DNA synthesis in phytochrome-mediated photomorphogenesis

In order to clarify the relationship between photomorphogenesis and DNA replication we investigated the effect of continuous far-red or white light on the synthesis of DNA in the cotyledons and the hypocotyl of mustard seedlings between 36 and 108 h after sowing. The total DNA content of the cotyledons (about 2.2 pg cell^-1) did not significantly change during this period although long-term labeling experiments revealed newly synthesized DNA of nuclear, plastid, and mitochondrial origin. Light had no detectable effect on total DNA content and on the labeling of either DNA fraction. Histoautoradiography indicated that nuclear DNA synthesis was exclusively localized in dividing stomatal cells and in sieve tube companion cells undergoing endopolyploidization. The DNA content of the hypocotyl increased continuously but likewise showed no detectable effect of light. It is concluded that cell growth and differentiation during photomorphogenesis is independent of DNA synthesis.Abbreviation DABA 3,5-diaminobenzoic acid     

Plastid-DNA levels in the different tissues of potato

The plastid-(pt) DNA levels in the different tissues of potato (Solanum tuberosum L.), including tubers of differing ages, have been studied. The DNA could be detected as a single nucleoid in amyloplasts of cells from young potato tubers by fluorescence microscopy, following staining of glutaraldehyde-fixed tissue with 4,6-diamidino-2-phenyl indole (DAPI). The renaturation kinetics of spinach ptDNA in the presence of total DNA from potato tissues and the fragments generated by restriction-enzyme digestion of potato-tuber DNA and chloroplast DNA indicated that the ptDNA of potato-tuber amyloplasts and of potato-leaf chloroplasts is essentially the same. Expressed as a percentage of the total DNA the level of ptDNA (5.2%) found in tubers, while less than that found in leaves (7.6%) was more than that found in petioles (3.4%), stems (3.0%) and roots (1.0%). There was a high level of both nuclear and plastid ploidy in mature potato-tuber cells and, on average, nuclei contained 32 pg of DNA (equivalent to 14C) and the 40 amyloplasts per cell contained DNA equivalent to 7800 copies of ptDNA, or 195 copies per amyloplast.Abbreviations DAPI 4,6-diamidino-2-phenyl-indole - LSU large sub-unit of ribulose-1,5-bisphosphate carboxylase - mtDNA mitochondrial DNA - ptDNA chloroplast or plastid DNA     

Excision and replication of extrachromosomal DNA of pea (Pisum sativum).

Experiments with cultured pea roots were conducted to determine (i) whether extrachromosomal DNA was produced by cells in the late S phase or in the G2 phase of the cell cycle, (ii) whether the maturation of nascent DNA replicated by these cells achieved chromosomal size, (iii) when extrachromosomal DNA was removed from the chromosomal duplex, and (iv) the replication of nascent chains by the extrachromosomal DNA after its release from the chromosomal duplex. Autoradiography and cytophotometry of cells of carbohydrate-starved root tips revealed that extrachromosomal DNA was produced by a small fraction of cells accumulated in the late S phase after they had replicated about 80% of their DNA. Velocity sedimentation of nascent chromosomal DNA in alkaline sucrose gradients indicated that the DNA of cells in the late S phase failed to achieve chromosomal size. After reaching sizes of 70 X 10(6) to 140 X 10(6) daltons, some of the nascent chromosomal molecules were broken, presumably releasing extrachromosomal DNA several hours later. Sedimentation of selectively extracted extrachromosomal DNA either from dividing cells or from those in the late S phase showed that it replicated two nascent chains, one of 3 X 10(6) daltons and another of 7 X 10(6) daltons. Larger molecules of extrachromosomal DNA were detectable after cells were labeled for 24 h. These two observations were compatible with the idea that the extrachromosomal DNA was first replicated as an integral part of the chromosomal duplex, was cut from the duplex, and then, once free of the chromosome, replicated two smaller chains of 3 X 10(6) and 7 X 10(6) daltons.     

Structural similarities between a mitochondrially encoded polypeptide and a family of prokaryotic respiratory toxins involved in plasmid maintenance suggest a novel mechanism for the evolutionary maintenance of mitochondrial DNA

Summary Subunit 8 of mitochondrial ATP synthase (A8), a mitochondrially encoded polypeptide, has no known homologue in any prokaryotic or plastid ATP synthase, suggesting that it has been recruited to its present role in the enzyme from an extraneous source. The polypeptide is poorly conserved at the primary sequence level, but shows a well-conserved hydropathy profile. The hydropathy profiles of A8 from diverse taxa were compared with those of thehok family of prokaryotic respiratory toxins, some of whose members are involved in plasmid maintenance, through postsegregational killing of cells that lose the plasmid at cell division. Such comparisons revealed a highly significant degree of similarity, suggesting a functional relationship. Based on these findings, it is proposed that A8 evolved from ahok-like protein, whose original role was the maintenance of an extrachromosomal replicon in the endosymbiont ancestor of mitochondria. An aggressive mechanism for the evolutionary maintenance of mitochondrial DNA overcomes many of the failings of traditional explanations for its retention as a separate genome.     

The genome of Eimeria spp., with special reference to Eimeria tenella--a coccidium from the chicken

Eimeria spp. contain at least four genomes. The nuclear genome is best studied in the avian species Eimeria tenella and comprises about 60 Mbp DNA contained within ca. 14 chromosomes; other avian and lupine species appear to possess a nuclear genome of similar size. In addition, sequence data and hybridisation studies have provided direct evidence for extrachromosomal mitochondrial and plastid DNA genomes, and double-stranded RNA segments have also been described. The unique phenotype of "precocious" development that characterises some selected lines of Eimeria spp. not only provides the basis for the first generation of live attenuated vaccines, but offers a significant entrée into studies on the regulation of an apicomplexan life-cycle. With a view to identifying loci implicated in the trait of precocious development, a genetic linkage map of the genome of E. tenella is being constructed in this laboratory from analyses of the inheritance of over 400 polymorphic DNA markers in the progeny of a cross between complementary drug-resistant and precocious parents. Other projects that impinge directly or indirectly on the genome and/or genetics of Eimeria spp. are currently in progress in several laboratories, and include the derivation of expressed sequence tag data and the development of ancillary technologies such as transfection techniques. No large-scale genomic DNA sequencing projects have been reported.     

NUPTs in sequenced eukaryotes and their genomic organization in relation to NUMTs

NUPTs (nuclear plastid DNA) derive from plastid-to-nucleus DNA transfer and exist in various plant species. Experimental data imply that the DNA transfer is an ongoing, highly frequent process, but for the interspecific diversity of NUPTs, no clear explanation exists. Here, an inventory of NUPTs in the four sequenced plastid-bearing species and their genomic organization is presented. Large genomes with a predicted low gene density contain more NUPTs. In Chlamydomonas and Plasmodium, DNA transfer occurred but was limited, probably because of the presence of only one plastid per cell. In Arabidopsis and rice, NUPTs are frequently organized as clusters. Tight clusters can contain both NUPTs and NUMTs (nuclear mitochondrial DNA), indicating that preNUPTs and preNUMTs might have concatamerized before integration. The composition of such a hypothetical preNUPT-preNUMT pool seems to be variable, as implied by substantially different NUPTs:NUMTs ratios in different species. Loose clusters can span several dozens of kbps of nuclear DNA, and they contain markedly more NUPTs or NUMTs than expected from a random genomic distribution of nuclear organellar DNA. The level of sequence similarity between NUPTs/NUMTs and plastid/mitochondrial DNA correlates with the size of the integrant. This implies that original insertions are large and decay over evolutionary time into smaller fragments with diverging sequences. We suggest that tight and loose clusters represent intermediates of this decay process.     

Whole Mitochondrial and Plastid Genome SNP Analysis of Nine Date Palm Cultivars Reveals Plastid Heteroplasmy and Close Phylogenetic Relationships among Cultivars

Date palm is a very important crop in western Asia and northern Africa, and it is the oldest domesticated fruit tree with archaeological records dating back 5000 years. The huge economic value of this crop has generated considerable interest in breeding programs to enhance production of dates. One of the major limitations of these efforts is the uncertainty regarding the number of date palm cultivars, which are currently based on fruit shape, size, color, and taste. Whole mitochondrial and plastid genome sequences were utilized to examine single nucleotide polymorphisms (SNPs) of date palms to evaluate the efficacy of this approach for molecular characterization of cultivars. Mitochondrial and plastid genomes of nine Saudi Arabian cultivars were sequenced. For each species about 60 million 100 bp paired-end reads were generated from total genomic DNA using the Illumina HiSeq 2000 platform. For each cultivar, sequences were aligned separately to the published date palm plastid and mitochondrial reference genomes, and SNPs were identified. The results identified cultivar-specific SNPs for eight of the nine cultivars. Two previous SNP analyses of mitochondrial and plastid genomes identified substantial intra-cultivar ( = intra-varietal) polymorphisms in organellar genomes but these studies did not properly take into account the fact that nearly half of the plastid genome has been integrated into the mitochondrial genome. Filtering all sequencing reads that mapped to both organellar genomes nearly eliminated mitochondrial heteroplasmy but all plastid SNPs remained heteroplasmic. This investigation provides valuable insights into how to deal with interorganellar DNA transfer in performing SNP analyses from total genomic DNA. The results confirm recent suggestions that plastid heteroplasmy is much more common than previously thought. Finally, low levels of sequence variation in plastid and mitochondrial genomes argue for using nuclear SNPs for molecular characterization of date palm cultivars.     

Senescence in the nongreening region of the rice (Oryza sativa) coleoptile

Summary The coleoptile of rice (Oryza sativa L. cv. Nippon-bare) emerges from the imbibed seed on day 2 after sowing and ceases its growth on day 3. In cross section, the cells near the outer epidermis turn into green between days 2 and 3, while those near the inner epidermis remain colorless. In this study, the complete process of the development in the nongreening cells in the coleoptile was examined by fluorescence and electron microscopy. Embryonic morphology on day 0 was rapidly converted into the differentiated greening or nongreening cells between days 1 and 2. Senescence in the inner, nongreening region first appeared on day 4 in the third or fourth cell layer from the inner epidermis and then spread towards both the inner and the outer epidermis, and the inner cells collapsed completely before the outer cells senesced. Cells adjacent to the inner epidermis, which senesced slowly, followed a sequence of events during development: (1) degradation of plastid DNA; (2) dispersal of nuclear chromatin, differentiation of plastids into amyloplasts, degradation of mitochondrial DNA; (3) degradation of the starch in amyloplasts; (4) disorganization of plastids; (5) condensation of the nucleus, shrinkage of mitochondria; (6) complete loss of cellular components, distortion of cell walls. In the interior cells, the early events including degeneration of plastid DNA and mitochondrial DNA occurred in parallel with those in the cells adjacent to the inner epidermis, yet rapid collapse of all the cellular components proceeded between days 3 and 5, and nuclear condensation could not be detected.Abbreviations cpDNA chloroplast DNA - DAPI 4,6-diamidino-2-phenylindole - DiOC₇ 3,3-dihexyloxacarbocyanine - IE inner epidermis - mtDNA mitochondrial DNA - mt-nucleoid mitochondrial nucleoid - OE outer epidermis - ptDNA plastid DNA - pt-nucleoid plastid nucleoid     

Chloroplast DNA content in Dinophysis (Dinophyceae) from different cell cycle stages is consistent with kleptoplasty

Kleptoplasty is the retention of plastids obtained from ingested algal prey, which can remain temporarily functional and be used for photosynthesis by the predator. With a new approach based on cell cycle analysis, we have addressed the question of whether the toxic, bloom-forming dinoflagellate Dinophysis norvegica practice kleptoplasty or if they replicate their own plastid DNA. Dividing (G2) and non-dividing (G1) D. norvegica cells from a natural population were physically separated with a flow cytometer based on their DNA content. Average numbers of nuclear and plastid rDNA copies were quantified with real-time PCR both in the G1 and G2 group. Cells from the G1 group contained 5800 ± 340 copies of nuclear rDNA and 1300 ± 200 copies of plastid rDNA; cells from the G2 group contained 9700 ± 58 copies of nuclear rDNA and 1400 ± 220 copies of plastid rDNA (mean ± SD, n  = 3). The ratio G2/G1 in average rDNA copies per cell was 1.67 for nuclear DNA and 1.07 for plastid DNA. These ratios show that plastid acquisition in D. norvegica is either uncoupled with the cell cycle, or plastids accumulate rapidly in the beginning of the cell cycle owing to feeding, as would be expected in a protist with kleptoplastic behaviour but not in a protist with own plastid replication. In addition, flow cytometry measurements on cells from the same population used for real-time PCR showed that when kept without plastidic prey, live Dinophysis cells lost on average 36% of their plastid phycoerythrin fluorescence in 24 h. Together these findings strongly suggest that D. norvegica does not possess the ability for plastid replication.     

Decrease in mitochondrial DNA and concurrent increase in plastid DNA in generative cells of Pharbitis nil during pollen development.

The amount of organellar DNA in a generative cell of Pharbitis nil was observed when squashed pollen grains collected on the day of flowering were stained with the DNA-specific fluorochrome 4',6-diamidino-2-phenylindole (DAPI). Using both DAPI-fluorescence microscopy and electron microscopy, observation of the same thin section of Technovit 7100 resin-embedded material revealed that all of the organellar DNA in mature generative cells is plastid DNA, and there is no mitochondrial DNA. During pollen development, we observed organellar DNA in fluorescence microscopic images using double-staining with DAPI and 3,3'-dihexyloxacarbocyanine iodide (DiOC6) and quantified the DNA using a video-intensified microscope photon counting system (VIMPCS). In the vegetative cells, the amounts of both mitochondrial and plastid DNA progressively decreased and had disappeared by 2 days before flowering. In the generative cells, mitochondrial DNA disappeared sooner than in the vegetative cells, indicating a more active mechanism for the decrease in mitochondrial DNA in the generative cells. In contrast, plastid DNA in the generative cells increased markedly. The DNA content per plastid was at a minimum value (corresponding to one copy of the plastid genome) 7 days before flowering, but it increased to a maximum value (corresponding to over 10 copies of the plastid genome) 2 days before flowering. Similar results were also obtained with immunogold electron microscopy using an anti-DNA antibody. These results suggest that the DNA content of mitochondria and plastids in P. nil is controlled independently during pollen development.     

Analysis of plastid and mitochondrial DNA insertions in the nucleus (NUPTs and NUMTs) of six plant species: size,relative age and chromosomal localization

We analysed the size, relative age and chromosomal localization of nuclear sequences of plastid and mitochondrial origin (NUPTs-nuclear plastid DNA and NUMTs-nuclear mitochondrial DNA) in six completely sequenced plant species. We found that the largest insertions showed lower divergence from organelle DNA than shorter insertions in all species, indicating their recent origin. The largest NUPT and NUMT insertions were localized in the vicinity of the centromeres in the small genomes of Arabidopsis and rice. They were also present in other chromosomal regions in the large genomes of soybean and maize. Localization of NUPTs and NUMTs correlated positively with distribution of transposable elements (TEs) in Arabidopsis and sorghum, negatively in grapevine and soybean, and did not correlate in rice or maize. We propose a model where new plastid and mitochondrial DNA sequences are inserted close to centromeres and are later fragmented by TE insertions and reshuffled away from the centromere or removed by ectopic recombination. The mode and tempo of TE dynamism determines the turnover of NUPTs and NUMTs resulting in their species-specific chromosomal distributions.     

A microspectrofluorometric analysis of nuclear and chloroplast DNA in Volvox

Nuclear division immediately follows nuclear DNA doubling in all stages of the life cycle examined in the green alga Volvox; fluorescence microfluorometry of individual cells revealed no evidence of prolonged accumulation of nuclear DNA prior to mitosis in reproductive cells. Somatic cell nuclear DNA quantity is unaffected by developmental events in gonidia of the same spheroid; it remains constant from the end of cleavage until the death of the cell. In reproductive cells, chloroplast DNA replication precedes nuclear replication. The sites of plastid DNA accumulation, made visible by use of the fluorochrome 4′,6-diamidino-2-phenylindole, increase in number during the prolonged growth phase of the V. carteri gonidium. Microspectrofluorometry of fluorochrome-stained DNA in situ shows that plastid DNA increases exponentially throughout this phase. The continuous plastid DNA accumulation during gonidial growth appears to represent a prokaryote-like instead of a eukaryote-like control of DNA synthesis. Most somatic cells contain plastid DNA, and this does not increase in amount during colony growth and reproduction. Most sperm cells also contain plastid DNA, although approximately 5% of somatic cells and up to 20% of sperm cells have no discernable plastid DNA. This is the second group of organisms in which DNA-free plastids have been observed.     

DNA damage in plant herbarium tissue

Dried plant herbarium specimens are potentially a valuable source of DNA. Efforts to obtain genetic information from this source are often hindered by an inability to obtain amplifiable DNA as herbarium DNA is typically highly degraded. DNA post-mortem damage may not only reduce the number of amplifiable template molecules, but may also lead to the generation of erroneous sequence information. A qualitative and quantitative assessment of DNA post-mortem damage is essential to determine the accuracy of molecular data from herbarium specimens. In this study we present an assessment of DNA damage as miscoding lesions in herbarium specimens using 454-sequencing of amplicons derived from plastid, mitochondrial, and nuclear DNA. In addition, we assess DNA degradation as a result of strand breaks and other types of polymerase non-bypassable damage by quantitative real-time PCR. Comparing four pairs of fresh and herbarium specimens of the same individuals we quantitatively assess post-mortem DNA damage, directly after specimen preparation, as well as after long-term herbarium storage. After specimen preparation we estimate the proportion of gene copy numbers of plastid, mitochondrial, and nuclear DNA to be 2.4-3.8% of fresh control DNA and 1.0-1.3% after long-term herbarium storage, indicating that nearly all DNA damage occurs on specimen preparation. In addition, there is no evidence of preferential degradation of organelle versus nuclear genomes. Increased levels of C→T/G→A transitions were observed in old herbarium plastid DNA, representing 21.8% of observed miscoding lesions. We interpret this type of post-mortem DNA damage-derived modification to have arisen from the hydrolytic deamination of cytosine during long-term herbarium storage. Our results suggest that reliable sequence data can be obtained from herbarium specimens.     

Evolutionary Capture of Viral and Plasmid DNA by Yeast Nuclear Chromosomes

A 10-kb region of the nuclear genome of the yeast Vanderwaltozyma polyspora contains an unusual cluster of five pseudogenes homologous to five different genes from yeast killer viruses, killer plasmids, the 2μm plasmid, and a Penicillium virus. By further database searches, we show that this phenomenon is not unique to V. polyspora but that about 40% of the sequenced genomes of Saccharomycotina species contain integrated copies of genes from DNA plasmids or RNA viruses. We propose the name NUPAVs (nuclear sequences of plasmid and viral origin) for these objects, by analogy to NUMTs (nuclear copies of mitochondrial DNA) and NUPTs (nuclear copies of plastid DNA, in plants) of organellar origin. Although most of the NUPAVs are pseudogenes, one intact and active gene that was formed in this way is the KHS1 chromosomal killer locus of Saccharomyces cerevisiae. We show that KHS1 is a NUPAV related to M2 killer virus double-stranded RNA. Many NUPAVs are located beside tRNA genes, and some contain sequences from a mixture of different extrachromosomal sources. We propose that NUPAVs are sequences that were captured by the nuclear genome during the repair of double-strand breaks that occurred during evolution and that some of their properties may be explained by repeated breakage at fragile chromosomal sites.It is well known that the nuclear genomes of most eukaryotes contain integrated fragments of organellar DNA called NUMTs (nuclear copies of mitochondrial DNA) and NUPTs (nuclear copies of plastid DNA, in plants) (26, 29, 44, 45, 57). These fragments are usually pseudogenes, although some NUMTs and NUPTs have become incorporated into functional nuclear genes (38). The NUMTs present in the nuclear genomes of Saccharomycotina yeast species were recently analyzed by Sacerdot et al. (48).In addition to their mitochondrial genomes, yeast species contain a variety of other extranuclear DNA and RNA elements, including viruses and plasmids. These extrachromosomal elements are usually considered to be autonomous entities that do not interact with nuclear DNA. When our laboratory sequenced the genome of the yeast Vanderwaltozyma polyspora (synonym: Kluyveromyces polysporus) (49), we were therefore surprised to find the genomic region we describe here, which contains integrated fragments of several plasmid- and virus-like sequences. We propose that this region was formed by the capture of plasmid and viral sequences by the same mechanism that captures mitochondrial DNA to form NUMTs (43, 65). In a literature search, we could find only one previous report of a similar finding: Utatsu et al. (59) reported the sequences of two regions of nuclear DNA from Zygosaccharomyces rouxii that were highly similar to parts of the 2μm-like plasmid pSR1 from that species, but rearranged.Before describing the V. polyspora region, and similar regions found in other species, we will first briefly introduce the extrachromosomal RNA and DNA entities that are known to exist in yeasts. Extrachromosomal nucleic acids are relatively uncommon in yeasts: a broad survey of 1,800 strains from 600 species by Fukuhara (14) found that 196 strains (11%) contained some sort of extrachromosomal entity. Among these, 105 strains had a double-stranded RNA (dsRNA), 28 had a linear dsDNA plasmid, and 53 had a circular DNA plasmid of the 2μm family. These elements typically also have a patchy distribution within a species, being found in some individuals or strains but not in others. For instance, Nakayashiki et al. (37) surveyed 70 “wild” strains of Saccharomyces (mostly S. cerevisiae) for the presence of five extrachromosomal elements (2μm DNA plasmid, L-A and L-BC helper RNA viruses, and W and T RNA entities) and found each element to be present in between 1 and 38 of the strains, with 1 strain even containing all five elements simultaneously.