Nuclear suppressors of the [poky] cytoplasmic mutant in Neurospora crassa. III. Effects on other cytoplasmic mutants and on mitochondrial ribosome assembly in [poky].

Summary We have previously isolated six non-allelic, nuclear mutations (su ^I loci) that partially suppress the growth, respiratory and cytochrome abnormalities of the extranuclear [poky] mutant.A comparison of the mitochondrial ribosome profiles of suppressed and unsuppressed [poky] strains revealed that five of the six suppressors alleviate at least partially the deficiency of mitochondrial small ribosomal subunits that is associated with the [poky] genotype.Six independently isolated Group I extranuclear mutants, namely [exn-1], [exn-2], [exn-4], [stp-B ₁], [SG-1] and [SG-3], which have growth and cytochrome phenotypes similar to [poky], also were found to be deficient in small subunits of mitochondrial ribosomes. Using cytochrome aa ₃ and b production as a criterion for mitochondrial protein synthesis, it could be shown that the nuclear su ^I suppressors of [poky] also suppress the other six Group I extranuclear mutants. However, differences in the efficiencies of suppression by su ^I suppressors suggest that at least some of Group I extrachromosomal mutants are not simply re-isolates of [poky], but represent distinct extranuclear mutations.     

The foundation of extranuclear inheritance: plastid and mitochondrial genetics

In 1909 two papers by Correns and by Baur published in volume 1 of Zeitschrift für induktive Abstammungs- und Vererbungslehre (now Molecular Genetics and Genomics) reported on the non-Mendelian inheritance of chlorophyll deficiencies. These papers, reporting the very first cases of extranuclear inheritance, laid the foundation for a new field: non-Mendelian or extranuclear genetics. Correns observed a purely maternal inheritance (in Mirabilis), whereas Baur found a biparental inheritance (in Pelargonium). Correns suspected the non-Mendelian factors in the cytoplasm, while Baur believed that the plastids carry these extranuclear factors. In the following years, Baur’s hypothesis was proved to be correct. Baur subsequently developed the theory of plastid inheritance. In many genera the plastids are transmitted only uniparentally by the mother, while in a few genera there is a biparental plastid inheritance. Commonly there is random sorting of plastids during ontogenetic development. Renner and Schwemmle as well as geneticists in other countries added additional details to this theory. Pioneering studies on mitochondrial inheritance in yeast started in 1949 in the group of Ephrussi and Slonimski; respiration-deficient cells (petites in yeast, poky in Neurospora) were demonstrated to be due to mitochondrial mutations. Electron microscopical and biochemical studies (1962–1964) showed that plastids and mitochondria contain organelle-specific DNA molecules. These findings laid the molecular basis for the two branches of extranuclear inheritance: plastid and mitochondrial genetics.     

Transmission and recombination of extranuclear genes during sexual crosses in Aspergillus nidulans

Summary Three extranuclear mitochondrial mutations in Aspergillus nidulans, (oliA1), (camA1) and (cs67), were used as markers in sexual crosses to provide information on the frequencies of transmission and recombination of the mitochondrial genome. Any individual perithecium contained ascospores of only one extranuclear genotype.Using mono-, bi- and trifactorial crosses it was found that all three markers could be recovered from the progeny, although the transmission frequencies were different for each marker. This bias was present irrespective of the nuclear background or the presence of selective agents in the medium on which the cross was established. These findings enable a series of transmission strength to be established, as shown below:- (cs67,{\text{ }}camA1) > ( + ) = (cs67) > (oliA1,cs67) \hfill \\ {\text{ }} > (oliA1) > (oliA1,{\text{ }}camA1) \hfill \\ \end{gathered} $$ " align="middle" border="0"> However, the numbers of recombinants isolated were so variable as to make this form of analysis unsuitable for mapping the mitochondrial genome.     

Recombination between the extranuclear genes conferring oligomycin resistance and cold sensitivity in Aspergillus nidulans

Summary Recombination has been demonstrated between the extranuclear loci (oliA1) and (cs67) of Aspergillus nidulans. The stability of the double mutant recombinant and the fact that it formed smaller colonies than either parent at the non-permissive temperature are strong evidence that physical recombination of the extranuclear DNA has occurred rather than simple mixing. A method has been developed for quantifying the extranuclear recombination frequency, thus providing a means of mapping the A. nidulans mitochondrial genome. The data obtained suggests that the two loci are not closely linked.     

Nuclear-extranuclear interactions affecting oligomycin resistance in Aspergillus nidulans.

Summary The extranuclear mitochondrial oligomycin-resistant mutation ofAspergillus nidulans, (oliA1), was transferred asexually into four nuclear oligomycin-resistant strains of different phenotypes. In all four cases, the possession of the nuclear plus extranuclear mutation led to an increase in the in vivo level of oligomycin resistance. In two cases, the altered cytochrome spectrum and impaired growth ability determined by (oliA1) were suppressed by the nuclear mutations. In the third case, the in vitro oligomycin resistance of the double mutant ATPase was dramatically increased above that of either of the component single mutant strains, indicating a synergystic interaction between the nuclear and extranuclear gene products. In the fourth case, the double mutant became cold-sensitive.A new extranuclear mitochondrial oligomycin-resistant mutation (oliB332) is described. This mutant is phenotypically similar to, though not identical with, (oliA1) but is separable by recombination.A range of nuclear oligomycin-resistant mutants have been mapped. Despite presenting five distinctly different phenotypes, they all map at the same locus.     

A novel extranuclear mutant of Neurospora with a temperature-sensitive defect in mitochondrial protein synthesis and mitochondrial ATPase

Summary [C93] is a novel, extranuclear mutant of Neurospora crassa which has a normal mitochondrial phenotype when grown at 25°, but which is deficient in cytochromes b and aa ₃ when grown at 37° (Pittenger and West 1979). In the present work, the phenotype of [C93] was characterized in greater detail. When [C93] is grown at 37°, the rate of mitochondrial protein synthesis is decreased to approximately 25% that of wild type; the ratio of mitochondrial small to large ribosomal subunits is decreased to 1:4 and mitochondrial small subunits are deficient in the mitochondrially-synthesized protein, S-5. The mitochondrial ribosome assembly defects in 37°-grown [C93] resemble those in chloramphenicol-treated wild-type cells and could merely be a consequence of the decreased rates of mitochondrial protein synthesis. Analysis of mitochondrial translation products by SDS gel electrophoresis suggests that 37°-grown [C93] is grossly deficient in the 19,000 Mr subunit of the oligomycin-sensitive ATPase relative to other mitochondrially-synthesized proteins. The ATPase defect was not found in other extranuclear or nuclear mutants deficient in mitochondrial protein synthesis. These data and additional evidence suggest that the primary defect in [C93] may be in the assembly of the ATPase complex. The possible connection between the ATPase defect and the deficiency of mitochondrial protein synthesis is discussed.     

Assembly-free quantification of vagrant DNA inserts

Inserts of DNA from extranuclear sources, such as organelles and microbes, are common in eukaryote nuclear genomes. However, sequence similarity between the nuclear and extranuclear DNA, and a history of multiple insertions, make the assembly of these regions challenging. Consequently, the number, sequence and location of these vagrant DNAs cannot be reliably inferred from the genome assemblies of most organisms. We introduce two statistical methods to estimate the abundance of nuclear inserts even in the absence of a nuclear genome assembly. The first (intercept method) only requires low-coverage (<1×) sequencing data, as commonly generated for population studies of organellar and ribosomal DNAs. The second method additionally requires that a subset of the individuals carry extranuclear DNA with diverged genotypes. We validated our intercept method using simulations and by re-estimating the frequency of human NUMTs (nuclear mitochondrial inserts). We then applied it to the grasshopper Podisma pedestris, exceptional for both its large genome size and reports of numerous NUMT inserts, estimating that NUMTs make up 0.056% of the nuclear genome, equivalent to >500 times the mitochondrial genome size. We also re-analysed a museomics data set of the parrot Psephotellus varius, obtaining an estimate of only 0.0043%, in line with reports from other species of bird. Our study demonstrates the utility of low-coverage high-throughput sequencing data for the quantification of nuclear vagrant DNAs. Beyond quantifying organellar inserts, these methods could also be used on endosymbiont-derived sequences. We provide an R implementation of our methods called “vagrantDNA” and code to simulate test data sets.     

Complementation among cytoplasmic mutants of Neurospora crassa

Summary Heteroplasmons with normal growth rates are formed when the slow-growing, female fertile, group I or II extranuclear mutants of Neurospora crassa are combined by forced heterokaryosis with the female sterile, stopper mutants of group III. Different mutants from the same growth and fertility group do not complement each other, and the poky-like strains of group I do not interact synergistically with [mi-3], the only known group II mutant. The mitochondrial cytochrome system of the complementing heteroplasmons are as abnormal as the cytochrome complements of the component extranuclear mutants, indicating that defects in the electron transport system represented by those mutants are related inconsequentially to growth. The observed functional complementation indicates the expression of the mitochondrial genome is not restricted to the specific organelle of which it is a part.Contribution No. 1255 Department of Agronomy; Contribution No. 1148, Division of Biology, Kansas Agriculture Experiment Station, Manhattan, Kansas.     

Biogenesis of mitochondria 44

Summary A comparative study of eight independently isolated mitochondrial oligomycin resistant mutants obtained from three laboratories show a variety of phenotypes based on cross resistance to venturicidin and sensitivity to low temperature. Analysis of recombination between pairs of markers indicate the existence of at least three genetic classes; class A, cross resistant to venturicidin and including the mutations O III, [oli1-r], [OLG1-R], [tso-r]; class B, mutations O _I, [oli17-r], [OLG2-R]; and class C, the mutation O _II. The recombination data is consistent with mutations of each class residing in three separate genes, although mutations of class A and B show very close linkage.Recombination in non-polar crosses has demonstrated that markers of all three classes are linked to the mik1 locus in the configuration (AB)-mik1-C. The mapping of this segment with respect to other markers of the mitochondrial genome and the order of classes A and B was established by analyses of co-retention frequencies of markers in primary petite isolates as well as by analysis of marker overlap of genetically and physically defined petite genomes. The unambiguous order ery1-A-B-mik1-C-par was obtained. DNA-DNA hybridization studies using mtDNA isolated from selected petites confirms this map and estimates the physical separation of markers. A reasonable correlation exists in this region of the genome between distances estimated physically by hybridization and genetically by frequency of recombination in non-polar crosses.It is postulated that the oligomycin-mikamycin linkage group represents a cluster of genes involved in determining a number of mitochondrial membrane proteins associated with the mitochondrial ATPase and respiratory complex III.This work was supported by the Australian Research Grants Committee, Project D65/15930     

Genetic studies on mitochondrially inherited mikamycin-resistance in Paramecium aurelia

Summary Mutants of Paramecium aurelia resistant to mikamycin were shown by microinjection experiments to be due to changes in the extranuclear, mitochondrial genetic system. These mutants were also resistant to a low concentration of erythromycin.Double mutants, resistant to a high level of erythromycin and to mikamycin, were obtained by a two-stage mutation-like process. Injection of mitochondria from these resulted in transformation to the double mutant type, irrespective of the selective medium used to obtain the transformants.     

Extranuclear recombination in Aspergillus nidulans: closely-linked multiple chloramphenicol- and oligomycin-resistance loci.

Summary A nuclear, chloramphenicol-sensitive mutant cas-1 has been isolated which is cross sensitive to a number of drugs, including oligomycin and cycloheximide. Approximately one-third of the chloramphenicol-resistant mutants isolated from mutagenized conidia of this strain were found to be extranuclear, and exhibited a variety of phenotypes. One of these mutants, designated (camB51), was slow growing on drug-free medium and recombined at low frequency with the previously described mutant (camA112) (Gunatilleke et al., 1975).The majority of extranuclear oligomycin-resistant mutants isolated from cas-1 were indistinguishable from (oliA1) (Rowlands and Turner, 1973). Two mutants, (oliB322) and (oliB332), with similar but not identical phenotypes to (oliA1), recombined with the latter at low frequency but not with each other, thus representing a new class of extranuclear mutants.     

Mitochondrial ribosome assembly in Neurospora crassa: mutants with defects in mitochondrial ribosome assembly.

Summary We have examined mitochondrial (mt) ribosome assembly and-function in five nuclear and six extranuclear mutants of Neurospora crassa which had previously been characterized as deficient in cytochromes b and aa ₃. All six extranuclear mutants showed phenotypes similar to that previously described for the extranuclear [poky] mutant: small subunit-deficient with 19 S rRNA rapidly degraded. The nuclear mutants have the following phenotypes: 297-24 is mt small subunit deficient with 19 S RNA rapidly degraded. 289-56 is mt small subunit deficient but contains normal ratios of 19 S to 25 S RNA in whole mitochondria. 289-67 and 299-9 show defects in the processing of 25 S RNA leading to accumulation of a large precursor RNA. 289-4 is deficient in large subunits although a substantial, but less than normal, amount of 25 S RNA is present in the mitochondria.The present work provides new insight into the phenotypes of mt small subunit-deficient mutants. Previous studies using chloramphenicol suggest that some defects in the assembly of mt small subunits may arise secondarily as a result of inhibition of mt protein synthesis (LaPolla and Lambowitz, 1977; Lambowitz et al., 1979). Three mutants (289-56, 289-67 and 299-9) appear to show such defects. These strains contain incomplete mt small subunits which sediment more slowly than normal and are deficient in at least two proteins, S-5 and S-9. Correlation of mutant phenotypes with rates of mt protein synthesis in the different strains suggests that mt protein synthesis must be decreased to less than one half of the wild-type rate before secondary defects in mt small subunit assembly are observed. This threshold value is much lower than that which leads to gross deficiencies of cytochromes b and aa ₃. Although several mutants have phenotypes suggestive of alterations in mt ribosomal proteins, no such alterations could be identified by two dimensional gel electrophoresis.     

Genetic diversity and phylogeography of Daphnia similoides sinensis located in the middle and lower reaches of the Yangtze River

Geographical patterns, climate, and environmental change have important influences on the distribution and spread of aquatic organisms. However, the relationships between the geographical pattern and phylogenetics of Daphnia as well as environmental change are not well known. The genetic diversity and phylogeography of seven D. similoides sinensis populations located in the middle and lower reaches of the Yangtze River were investigated based on the combination of mitochondrial (COI gene) and nuclear (14 microsatellite primers) markers. Based on the mitochondrial gene markers, D. similoides sinensis from the middle and lower reaches of the Yangtze River had one ancestral haplotype and two evolutionary clades. In addition, D. similoides sinensis population deviated from neutral evolution, showing signs of a bottleneck effect followed by population expansion. Based on the microsatellite markers, the seven D. similoides sinensis populations formed three main groups. The dendrogram (NJ/ME) showed that D. similoides sinensis based on the mitochondrial genes marker were obviously clustered two main clades, whereas there were three clades based on the microsatellite markers. Our results suggested that the habitat fragmentation due to the barrier of the dams and sluices promoted the genetic differentiation and phylogeography of D. similoides sinensis populations in the middle and lower reaches of the Yangtze River.     

Genetic variation and evolutionary origins of parthenogenetic Artemia (Crustacea: Anostraca) with different ploidies

Using two nuclear (ITS1 and Na⁺/K⁺ ATPase) and three mitochondrial (COI, 16S and 12S) markers, we determined the genetic variation and evolutionary relationship of parthenogenetic and bisexual Artemia. Our analyses revealed that mitochondrial genes had higher genetic variation than nuclear genes and that the 16S showed more variety than the other mitochondrial genes in parthenogenetic populations. Triploid parthenogens showed lower genetic variation than diploid ones, whereas the tetra‐ and pentaploids had greater genetic distance than diploid parthenogens. No shared haplotype was found between individuals of parthenogenetic populations and Asian bisexual species with the exception of Na⁺/K⁺ ATPase (Artemia tibetiana). Only mitochondrial markers can demonstrate phylogenetic relationships, and showed that the parthenogenetic Artemia is a polyphyletic group in which the diploid lineages share a common ancestor with Artemia urmiana while tetraploids are closely related to Artemia sinica. The triploid and pentaploid linages are likely to be directly derived from diploid and tetraploid parthenogens, respectively. Subsequently, west Asia is origin for di‐/triploids, and tetra‐/pentaploids rose from East Asia.     

Widespread co‐occurrence of divergent mitochondrial haplotype lineages in a Central American species of poison frog (Oophaga pumilio)

Aim To analyse the phylogeographic structure of the strawberry poison frog, Oophaga pumilio (Dendrobatidae), across a large part of its range using a combination of mitochondrial and nuclear markers. Location Costa Rica and Panama. Methods Sequence analyses of a mitochondrial (cytochrome b) and a nuclear (RAG‐1) gene fragment as well as analyses of seven microsatellite loci were carried out on 269 individuals of O. pumilio sampled from 24 localities and on two individuals of O. vicentei. Results Two main mitochondrial haplotype lineages, corresponding to a northern (north Costa Rica) and a southern (south Costa Rica and eastern Panama) lineage, were identified. They differed by up to 7% uncorrected distance. We observed co‐occurrence of both lineages in seven populations in Costa Rica and Panama, indicating a pattern of extensive admixture. The two main mitochondrial lineages of O. pumilio roughly corresponded to a previously described phylogeographic pattern. Microsatellites indicate admixture spanning over a wide geographic area, but significant variation between the northern and southern groups was also found with microsatellite data. While microsatellite data reconstructed a separation south of an assumed Caribbean valley barrier, mitochondrial haplotypes of the ‘southern lineage’ shifted this barrier towards the north. Main conclusions Despite admixture, all three markers showed significant variation between the northern and southern groups. Phylogeographical breaks known from other anuran species in the study region could not be verified for O. pumilio. The unexpected clustering of the population from Escudo de Veraguas and the individuals of O. vincentei with the northern O. pumilio lineage indicates the need for a fundamental and careful taxonomic revision, including an interspecific phylogeography of the entire genus.     

Localization of a non-Mendelian gene in Chlamydomonas reinhardii

Summary Transfer of a non-Mendelian neamine-dependent (nd) mutant to an antibioticfree medium results in neamine-sensitive and neamine-resistent revertants. These reversions are caused by extranuclear mutations.The neamine-sensitive revertants are no more able to split offnd-cells after back-donation to neamine containing medium. Therefore they are different from the streptomycin-sensitive revertants of a streptomycin-dependent (sd) mutant. These mutants were capable ofsd-segregation though their potence ofsd-segregation diminished on antibiotic-free medium with increasing time of cultivation.The different behaviour can be explained by the fact that manysd-genes are present which have to be appointed to the mitochondria. On the other side, thend-gene exists only in few copies and is located therefore in the chloroplast.Several experiments with differing methods are discussed to localize the extranuclear genes.

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Vorgelegt durch G. Melchers     

Identification of sterility-inducing cytoplasms in rye using the plasmotype–genotype interaction test and newly developed SCAR markers

A series of 25 rye (Secale cereale L.) inbred lines was tested with respect to three mitochondrial sequence-characterized amplified region (SCAR) polymorphisms. The analysis revealed a close association between the marker-determined mitotypes and plasmotypes (cytoplasm types known from breeding data) represented by the inbreds. The mitochondrial markers also confirmed normal (N-) cytoplasmic character of three wild rye species: Secale montanum, S. vavilovii and S. kuprijanovii. For 186 plants from open-pollinated cultivars of Turkish and South American origin, cytoplasm identification was performed with the use of crossing with double non-restoring tester. In 77 plants the normal (N) cytoplasm was detected, and for 63 of these the PCR analysis was performed producing results which were consistent with the genetic data based on testcrossing and phenotype assessment. The mitochondrial markers also confirmed a presence of sterility-inducing cytoplasm in the remaining 109 plants. Moreover, the markers allowed for differentiation between Pampa (P-) and Vavilovii (V-) cytoplasmic individuals. For 60 plants the latter results were verified using crosses with a line maintaining P-cytoplasmic sterility and acting as a restorer of the V-cytoplasm. For two of these plants contradicting results were produced with the applied methods of cytoplasm identification and the basis of this discrepancy is discussed. Regardless of the identification method, widespread occurrence of a sterility-inducing cytoplasm was revealed, especially in South American populations.     

NUCLEAR AND MITOCHONDRIAL SEQUENCE DATA REVEAL AND CONCEAL DIFFERENT DEMOGRAPHIC HISTORIES AND POPULATION GENETIC PROCESSES IN CARIBBEAN REEF FISHES

Mitochondrial and nuclear sequence data should recover historical demographic events at different temporal scales due to differences in their effective population sizes and substitution rates. This expectation was tested for two closely related coral reef fish, the tube blennies Acanthemblemaria aspera and A. spinosa. These two have similar life histories and dispersal potentials, and co‐occur throughout the Caribbean. Sequence data for one mitochondrial and two nuclear markers were collected for 168 individuals across the species’ Caribbean ranges. Although both species shared a similar pattern of genetic subdivision, A. spinosa had 20–25 times greater nucleotide sequence divergence among populations than A. aspera at all three markers. Substitution rates estimated using a relaxed clock approach revealed that mitochondrial COI is evolving at 11.2% pairwise sequence divergence per million years. This rapid mitochondrial rate had obscured the signal of old population expansions for both species, which were only recovered using the more slowly evolving nuclear markers. However, the rapid COI rate allowed the recovery of a recent expansion in A. aspera corresponding to a period of increased habitat availability. Only by combining both nuclear and mitochondrial data were we able to recover the complex demographic history of these fish.     

Development of a high-resolution molecular marker for tracking Pseudo-nitzschia pungens genetic diversity through comparative analysis of mitochondrial genomes

The harmful algal bloom (HAB) species Pseudo-nitzschia pungens is widely distributed in almost all continents. Accumulating evidence suggests that P. pungens has high genetic diversity and many strains can produce the toxin domoic acid (DA) that harms animals and humans. Nevertheless, different P. pungens strains cannot be distinguished using morphological features or using common molecular markers including 18S rDNA, 28S rDNA, ITS, cox1, and rbcL. As such, high-resolution molecular markers need to be developed to resolve P. pungens genetic diversity, facilitating accurate tracking of toxic P. pungens strains. We hypothesized that molecular markers with high resolution and high specificity can be designed through identifying regions with high genomic variations in the mitochondrial genome. Here, we describe the development of a new molecular marker Pseudo-nitzschia pungens mitochondrial 1 (ppmt1) with high resolution and high specificity through comparative analysis of mitochondrial genomes of nine P. pungens strains isolated from coastal regions of China. In conclusion, we have developed ppmt1 as a high-resolution and high-specificity molecular marker for tracking strains and genetic diversity of the HAB species P. pungens.

     

Phylogeography of the marine macroalga Sargassum hemiphyllum (Phaeophyceae,Heterokontophyta) in northwestern Pacific

Sargassum hemiphyllum is commonly found in Japan and Korea, with a variety, var. chinense, that is found distributed in the southern Chinese coast. We previously reported distinct genetic differentiation between the two taxa based on the PCR‐RFLP data of plastid RubiscoL‐S spacer. The present study aims at elucidating the phylogeographic pattern of S. hemiphyllum based on more markers in the nuclear and extranuclear genomes, with a view to reveal the occurrence of hybridization. The two allopatrically distributed taxa were found to be genetically distinct in nuclear ITS2, plastidial Rubisco (Rbc) and mitochondrial TrnW_I (Trn) spacers. Their divergence was postulated to be attributable to the vicariant event which resulted from the isolation of the Sea of Japan during the late Miocene (6.58–11.25 Mya). Divergence within both S. hemiphyllum and the chinense variety was observed based on Trn spacer, while the divergence in S. hemiphyllum was further confirmed in Rbc spacer. This divergence appears to correspond to the separation of the Japanese populations between the Sea of Japan and the Pacific that occurred around 0.92–2.88 Mya (the early Pleistocene). The presence of an ITS2 clone resembling var. chinense sequences in a Japanese population of S. hemiphyllum (JpNS) raises the possibility of the introgression of var. chinense individuals into S. hemiphyllum population. Compared to that between S. hemiphyllum and the chinense variety, hybridization among the Japanese and Korean populations of S. hemiphyllum is highly probable as all these individuals share a pool of nuclear ITS2 sequences, possibly attributable to incomplete concerted evolution of ITS2.