Intergenomic recombination of mitochondrial genomes in a somatic hybrid plant

Summary Restriction site polymorphisms between two parental mitochondrial (mt) genomes were used to characterize the novel mt genome present in a somatic hybrid plant. A cosmid clone containing mtDNA restriction fragments characteristic of both parental plant lines was identified in a library constructed from mtDNA of progeny of a somatic hybrid plant. Restriction mapping revealed the location of several restriction fragments derived from each of the parental mt genomes on the same cloned region of somatic hybrid mtDNA. This result is direct evidence that intergenomic recombination at the molecular level occurs in homologous regions of two parental mt genomes combined in the same plant cell by protoplast fusion.     

Characterization of mitochondrial DNA in chloramphenicol-resistant interspecific hybrids and a cybrid

We have examined the restriction endonuclease cleavage patterns exhibited by the mitochondrial DNAs (mtDNA) of four chloramphenicolresistant (CAP^R)human × mouse hybrids and one CAP^R cybrid derived from CAP^R HeLa cells and CAP^S mouse RAG cells. Restriction fragments of mtDNAs were separated by electrophoresis and transferred by the Southern technique to diazobenzyloxymethyl paper. The covalently bound DNA fragments were hybridized initially with ³² P-labeled complementary RNA (cRNA) prepared from human mtDNA and, after removal of the human probe, hybridized with mouse [³²P]cRNA prepared from mouse mtDNA. Three hybrids which preferentially segregated human chromosomes and the cybrid exhibited mtDNA fragments indistinguishable from mouse cells. One hybrid, ROH8A, which exhibited reverse chromosome segregation, contained only human mtDNA. The pattern of chromosome and mtDNA segregation observed in these hybrids and the cybrid support the hypothesis that a complete set of human chromosomes must be retained if a human mouse hybrid is to retain human mitochondrial DNA.     

A restriction endonuclease map of the chloroplast genome of pearl millet

Chloroplast DNA from pearl millet (Pennisetum americanum) was used to construct recombinant plasmids. These plasmids contained 97 kilobase pairs of unique DNA sequences. The chloroplast DNA fragments in these plasmids were mapped with the restriction endonucleases SalI, SphI, XhoI, BglI and HpaI. The technique of overlapping hybridization or chromosome walking was used to orient these DNA fragments on a restriction endonuclease map of the chloroplast genome. The size of the chloroplast DNA from pearl millet was estimated in this fashion to be 127–138 kilobase pairs. Twenty one kilobase pairs of the cloned DNA fragments were represented twice on the genome as inverted repeats. Thus, the recombinant plasmids which were isolated contained approximately 86–93% of the nucleotide sequences in the chloroplast genome of pearl millet. Previously characterized cloned chloroplast DNA sequences from other plants were used as hybridization probes to locate the genes for the large subunit of ribulose 1,5-bisphosphate carboxylase, the -coupling factor of ATPase and the 32 kilodalton polypeptide of photo system 11 on the restriction endonuclease map of the pearl millet chloroplast genome.     

Mitotic segregation of mitochondrial dnas in human cell hybrids and expression of chloramphenicol resistance

The relationship between the chloramphenicol (CAP)-resistant phenotype and the mtDNA genotype was investigated in segregating human, HeLa × HT1080, somatic cell hybrids. The parental mtDNAs were quantitated in heteroplasmic cells by using restriction fragment length polymorphisms (RFLPs) detected in Southern blots. CAP-resistant (R) × CAP-sensitive (S) hybrids selected and grown in CAP for brief periods had as little as 25% CAP-R mtDNA. With prolonged selection, the CAP-R mtDNA increased to 90–95%. Hybrids selected and passaged without CAP either retained both mtDNAs or progressively lost one mtDNA (mitotic segregation). The CAP-resistance phenotype of these hybrids changed abruptly when the proportion of CAP-R mtDNAs fluctuated around approximately 10% (threshold effect). Hybrids with greater than 25% HT1080 mtDNA had an additional characteristic. They cloned better with CAP than without. The cloning efficiency in CAP of hybrids having 90% HT1080 mtDNA was more than fivefold greater than the control.     

The chloroplast and mitochondrial DNA type are correlated with the nuclear composition of somatic hybrid calli of Solanum tuberosum and Nicotiana plumbaginfolia

This paper describes the analysis of chloroplast (cp) DNA and mitochondrial (mt) DNA in 21 somatic hybrid calli of Solanum tuberosum and Nicotiana plumbaginifolia by means of Southern-blot hybridization. Each of these calli contained only one type of cpDNA; 14 had the N. plumbaginifolia (Np) type and seven the S. tuberosum (St) type. N. plumbaginifolia cpDNA was present in hybrids previously shown to contain predominantly N. plumbaginifolia chromosomes whereas hybrids in which S. tuberosum chromosomes predominated possessed cpDNA from potato. We have analyzed the mtDNA of these 21 somatic hybrid calli using four restriction enzyme/probe combinations. Most fusion products had only, or mostly, mtDNA fragments from the parent that predominated in the nucleus. The hybrids containing mtDNA fragments from only one parent (and new fragments) also possessed chloroplasts from the same species. The results suggest the existence of a strong nucleo-cytoplasmic incongruity which affects the genome composition of somatic hybrids between distantly related species.     

Recombination of mitochondrial DNA without selection pressure among compatible strains of the Aspergillus niger species aggregate

Previous mitochondrial transmission experiments between oligomycin-resistant and oligomycin-sensitive incompatible strains of the A. niger aggregate bearing various mtDNA RFLP profiles resulted in a great variety of mitochondrial recombinants under selection pressure. Apart from the recombinant mtDNAs, resistant clones harbouring unchanged RFLP profiles of resistant donor mtDNAs with the recipient nuclear backgrounds were rarely isolated. These strains were anastomosed with nuclearly isogenic oligomycin-sensitive recipient partners and the mitochondria of the resulting progeny were examined under non-selective conditions. These experiments provide insights into events which are possibly similar to those occurring in nature. The heterokaryons obtained formed both oligomycin-resistant and -sensitive sectors, most of which were found to be homoplasmons. Progenies harbouring oligomycin-resistant and -sensitive mtDNAs may originate either from individual recombination events or be due to parental segregation. MtDNA recombination might take place in the heterokaryons without selection by oligomycin. The most frequent recombinant types of mtDNA RFLP profiles were indistinguishable from those recombinant mtDNAs which were frequently obtained under selection pressure from directed transfer experiments between incompatible strains. We present evidence that mixed mitochondrial populations may influence the compatibility reactions in the presence of an isogenic nuclear background, that recombination may take place without selection pressure, and that the process does not require specific nuclear sequences of both parental strains. Received: 20 October 1997 / 13 January 1998     

Ethidium bromide rejuvenation of senescent cultures of Podospora anserina : Loss of senescence-specific DNA and recovery of normal mitochondrial DNA

Summary The effect of ethidium bromide (EB) which is known to be able to rejuvenate senescent mycelia in Podospora anserina, has been investigated at the level of the mitochondrial DNA (mtDNA) by restriction analysis and molecular hybridization. While senescent mycelia display a very low growth ability and gross mtDNA modifications (tandem amplification of short sequences and disorganization of the mitochondrial chromosome: deletion of large sequences), the rejuvenated mycelia display a normal life span and contain a mtDNA in all respects identical to that of wild type mycelium (neither circular molecules nor amplified fragments could be detected). These results demonstrate a strict correlation between the senescent state and the presence of amplified mtDNA and suggest that EB rejuvenation could proceed by an efficient selection of intact mitochondrial chromosomes still present in senescent cultures.     

Mitochondrial DNA inheritance in sexual crosses of Pleurotus ostreatus

The inheritance of mitochondrial DNA (mtDNA) in sexual crosses was investigated to expand our understanding of the large genetic divergence in mtDNAs among natural populations of the higher basidiomycete Pleurotus ostreatus. Reciprocal crosses were made between compatible monokaryons with distinguishable mtDNA restriction fragment length polymorphisms (PFLPs). Almost all of the dikaryons produced by these crosses had mtDNA genotypes from one of the parental monokaryons. However, for dikaryons isolated from the junction-zone of crossed monokaryons, recombinant mitochondrial genomes commonly appeared. These results showed that P. ostreatus mtDNA can be inherited biparentally, via mtDNA recombination, as well as uniparentally. Further, it was suggested that mtDNA recombination may be an important source of variation in mitochondrial genomes among natural populations of P. ostreatus. Received: 4 June / 14 August 1996     

The <Emphasis Type="Italic">rpl5</Emphasis>–<Emphasis Type="Italic">rps14</Emphasis> mitochondrial region: a hot spot for DNA rearrangements in <Emphasis Type="Italic">Solanum</Emphasis> spp. somatic hybrids

Southern analysis with rpl5 and rps14 mtDNA gene probes of Solanum tuberosum, S. commersonii and a sample of somatic hybrids detected polymorphisms between parents and the appearance of a novel restriction fragment in various hybrids. In one of them, detailed mtDNA analyses revealed various configurations of the rpl5–rps14 region present at different stoichiometries. Multiple inter-parental recombination events across homologous sequences were assumed to have caused these rearrangements. Sequence similarity searches detected one sequence putatively involved in the recombination upstream of the rpl5 gene. The presence of a second recombinogenic sequence was inferred. We propose two models to explain the mechanism responsible for obtaining the different rpl5–rps14 arrangements shown after somatic hybridization. Variability in the rpl5–rps14 region observed in both the parental species and their somatic hybrids suggests this region is a hot spot for mtDNA rearrangements in Solanum spp.Contribution no. 39 from the Institute of Plant Genetics, Research Division of Portici.Communicated by A. Brennicke     

Comparative analysis of the mitochondrial genomes of Chlamydomonas eugametos and Chlamydomonas moewusii

Summary We report the cloning and physical mapping of the mitochondrial genome of Chlamydomonas eugametos together with a comparison of the overall sequence structure of this DNA with the mitochondrial genome of Chlamydomonas moewusii, its closely related and interfertile relative. The C. eugametos mitochondrial DNA (mtDNA) has a 24 kb circular map and is thus 2 kb larger than the 22 kb circular mitochondrial genome of C. moewusii. Restriction mapping and heterologous fragment hybridization experiments indicate that the C. eugametos and C. moewusii mtDNAs are colinear. Nine cross-hybridizing restriction fragments common to the C. eugametos and C. moewusii mtDNAs, and spanning the entirety of these genomes, show length differences between homologous fragments which vary from 0.1 to 2.3 kb. A 600 bp subfragment of C. moewusii mtDNA, within one of these conserved fragments, showed no hybridization with the C. eugametos mtDNA. Of the 73 restriction sites identified in the C. eugametos and C. moewusii mtDNAs, five are specific to C. moewusii, eight are specific to C. eugametos and 30 are common to both species. Hybridization experiments with gene probes derived from protein-coding and ribosomal RNA-coding regions of wheat and Chlamydomonas reinhardtii mtDNAs support the view that the small and large subunit ribosomal RNA-coding regions of the C. eugametos and C. moewusii mtDNAs are interrupted and interspersed with each other and with protein-coding regions, as are the ribosomal RNA-coding regions of C. reinhardtii mtDNA; however, the specific arrangement of these coding elements in the C. eugametos and C. moewusii mtDNAs appears different from that of C. reinhardtii mtDNA.     

Physical and genetic map of the mitochondrial genome of Cryphonectria parasitica Ep155

 In the chestnut-blight fungus, Cryphonectria parasitica, a cytoplasmically transmissible (infectious) form of hypovirulence is associated with mitochondrial DNA (mtDNA) mutations that cause respiratory deficiencies. To facilitate the characterization of such mutations, a restriction map including the probable location of 13 genes was constructed for a relatively well-characterized virulent strain of the fungus, Ep155. The physical map is based on the order of all fragments generated by cleavage of the mtDNA by the PstI restriction endonuclease and includes some of the cleavage sites for HindIII, EcoRI, and XbaI. It was constructed from hybridization patterns of cloned mtDNA fragments with Southern blots of mtDNA digested with the four restriction enzymes. On this map, the probable locations of genes commonly found in the mitochondrial genomes of ascomycetes were determined by low-stringency hybridization of cloned Neurospora crassa mitochondrial gene probes to Southern blots of C. parasitica mtDNA. The data indicate that the mtDNA of strain Ep155 is a circular molecule of approximately 157 kbp and ranks among the largest mitochondrial chromosomes observed so far in fungi. The mtDNAs of 11 different C. parasitica isolates range in size from 135 to 157 kbp and in relatedness from 68 to 100 percent, as estimated from restriction-fragment polymorphisms. In addition to the typical mtDNA, the mitochondria of some isolates of the fungus contain double-stranded DNA plasmids consisting of nucleotide sequences not represented in the mtDNA of Ep155. Received: 19 September 1995/4 January 1996     

Recombination between parental mitochondrial DNA following protoplast fusion can occur in a region which normally does not undergo intragenomic recombination in parental plants

Summary A novel restriction fragment, which was not present in either parent's mitochondrial DNA, was cloned from the mitochondrial genome of a somatic hybrid Petunia line. This fragment resulted from interspecific recombination between homologous mitochondrial DNA regions of the parental plants, Petunia line 3704 and line 3688. Hybridization with a cloned Petunia atp9 gene revealed that the regions involved in recombination carry atp9 coding sequences. Intragenomic recombination within the parental mitochondrial genomes was not detected between the atp9 regions which recombined following protoplast fusion.     

Structural and functional mitochondrial abnormalities associated with high levels of partially deleted mitochondrial DNAs in somatic cell hybrids

Kearns-Sayre syndrome (KSS) is a progressive and ultimately fatal human encephalomyopathy that is associated with large-scale deletions of mitochondrial DNA (mtDNA). To gain new insights into the developmental pathobiology of this disease, we studied the maintenance and expression of deleted mtDNAs (-mtDNAs) in somatic cell hybrids generated by fusion of HeLacot cells with a KSS fibroblast clone containing both wild-type and -mtDNAs. We observed that -mtDNAs were preferentially maintained over the KSS wild-type mtDNAs (wt-mtDNAs) in almost all isolated hybrid clones. Mitochondrial metabolism was not compromised in hybrids containing as much as 70–79% -mtDNAs. Two clones containing more than 99% -mtDNA were severely deficient in oxidative phosphorylation and exhibited abnormal, enlarged mitochondria. These clones had undetectable levels of mtDNA-encoded polypeptides, but contained normal amounts of a nuclear DNA-encoded mitochondrial protein. The data suggest a nonrandom pattern of mtDNA segregation in the triplasmic hybrids and a correlation among -mtDNA, structural mitochondrial abnormalities, and mitochondrial dysfunction.     

Tissue-specific genome instability in synthetic interspecific hybrids of <Emphasis Type="Italic">Pennisetum purpureum</Emphasis> (Napier grass) and <Emphasis Type="Italic">Pennisetum glaucum</Emphasis> (pearl millet) is caused by micronucleation

Genome instability is observed in several species hybrids. We studied the mechanisms underlying the genome instability in hexaploid hybrids of Napier grass (Pennisetum purpureum R.) and pearl millet (Pennisetum glaucum L.) using a combination of different methods. Chromosomes of both parental genomes are lost by micronucleation. Our analysis suggests that genome instability occurs preferentially in meristematic root tissue of hexaploid hybrids, and chromosome elimination is not only caused by centromere inactivation. Likely, beside centromere dysfunction, unrepaired DNA double-strand breaks result in fragmented chromosomes in synthetic hybrids.     

Mitochondrial DNA analysis of mouse-rat hybrid cells: Effect of chloramphenicol selection on the relative amounts of parental mitochondrial DNAs

Several mouse-rat somatic hybrid cell lines were isolated by fusing chloramphenicol-resistant (CAP^R) and CAP-sensitive (CAP^S) parent cells, and propagation of the parent mitochondrial DNA (mtDNA) species in the hybrid cells was studied. The restriction endonucleases EcoRI, HpaII, and HaeIII were used for identification of mtDNA species. Both mouse and rat mtDNAs were propagated in all the hybrid cells examined and maintained during long-term cultivation and repeated cell division. Moreover, in CAP^R mouse-rat hybrid cells, selection and successive cultivation in the presence of CAP did not increase the relative amount of mtDNA species of CAP^R parent cell origin, and when CAP was removed from the culture medium, mtDNA species of CAP^R parent cell origin did not decrease appreciably. The amount of mouse mtDNAs was consistently 1–4 times that of rat mtDNAs in the mouse-rat hybrid cells regardless of the species of parent cells from which the CAP resistance was derived. Thus mouse-rat hybrid cells have a stable mtDNA population in which the amount of mouse mtDNAs is larger than that of rat mtDNAs without any influence of CAP selection.     

Biased organelle transmission in somatic hybrids ofLycopersicon esculentum andSolanum lycopersicoides

Summary Restriction fragment length polymorphisms (RFLPs) were used to determine the transmission of organelle genomes in somatic hybrid plants of tomato and its wild relativeSolanum lycopersicoides. Biased frequencies of organelle combinations were observed in a population of 70 somatic hybrid plants each derived from a separate callus. The plastids in 68 of 70 hybrids examined were fromL. esculentum. One of the remaining hybrids, plant 240, hadS. lycopersicoides plastids and the other, plant 63, had a mixture of parental plastids. Forty-six of the same 70 plants were analyzed for mtDNA and all had that ofS. lycopersicoides including plant 240. One of these hybrids had novel mtDNA fragments which mayhave resulted from recombination or rearrangement. The biased transmission may have resulted from an initial unequal input of organelles, differential replication of organelles, or nucleo-organelle incompatibility.Michigan Agricultural Experiment Station Journal Article No. 12538. Supported by Grant No. I-751-84R from BARD, The United States-Israel Binational Agricultural Research and Development Fund     

Randomly amplified polymorphic DNAs assess recombination following an induced parasexual cycle in Penicillium roqueforti

Random amplified polymorphic DNAs (RAPDs) were used as a genetic marker system to characterize recombinant strains following the parasexual cycle of Penicillium roqueforti. After protoplast fusion and haploidization of diploid hybrids, segregants characterized by a reassortment of the parental genetic markers displayed specific RAPD fingerprints. The appearance or the loss of RAPD fragments demonstrate that these markers provide an efficient method to analyze recombination and to characterize somatic hybrids.     

Assignment of human gamma crystallin multigene family to chromosome 2

The multigene family for human -crystallin has been assigned to chromosome 2 using rodent-human somatic cell hybrids and filter hybridization analysis of cell hybrid DNA. Two genomic DNA probes containing human -crystallin gene sequences hybridize to five fragments in human DNA digested with the restriction enzyme EcoRI. By correlating the presence of these fragments in somatic cell hybrid DNA with the human chromosome content of the hybrids, at least six human -crystallin genes can be mapped to chromosome 2. Data obtained with a hybrid clone containing a mouse-human interspecies translocation suggest that these genes may be clustered together on the long arm of human chromosome 2.     

Location,identity, amount and serial entry of chloroplast DNA sequences in crucifer mitochondrial DNAs

Southern blot hybridization techniques were used to examine the chloroplast DNA (cpDNA) sequences present in the mitochondrial DNAs (mtDNAs) of two Brassica species (B. campestris and B. hirta), two closely related species belonging to the same tribe as Brassica (Raphanus sativa, Crambe abyssinica), and two more distantly related species of crucifers (Arabidopsis thaliana, Capsella bursa-pastoris). The two Brassica species and R. sativa contain roughly equal amounts (12–14 kb) of cpDNA sequences integrated within their 208–242 kb mtDNAs. Furthermore, the 11 identified regions of transferred DNA, which include the 5 end of the chloroplast psaA gene and the central segment of rpoB, have the same mtDNA locations in these three species. Crambe abyssinica mtDNA has the same complement of cpDNA sequences, plus an additional major region of cpDNA sequence similarity which includes the 16S rRNA gene. Therefore, except for the more recently arrived 16S rRNA gene, all of these cpDNA sequences appear to have entered the mitochondrial genome in the common ancestor of these three genera. The mitochondrial genomes of A. thaliana and Capsella bursa-pastoris contain significantly less cpDNA (5–7 kb) than the four other mtDNAs. However, certain cpDNA sequences, including the central portion of the rbcL gene and the 3 end of the psaA gene, are shared by all six crucifer mtDNAs and appear to have been transferred in a common ancestor of the crucifer family over 30 million years ago. 1n conclusion, DNA has been transferred sequentially from the chloroplast to the mitochondrion during crucifer evolution and these cpDNA sequences can persist in the mitochondrial genome over long periods of evolutionary time.