Biased inheritance of optional insertions following mitochondrial genome recombination in the basidiomycete fungus Coprinus cinereuss

Summary Two mitochondrial genomes of Coprinus cinereus, H and J, were found to have alternative 1.23 kb insertions. Using the Neurospora crassa cytochrome oxidase-1 (co-1) gene as a probe, the J insertion site was shown to be located within the Coprinus co-1 gene, whereas the H insertion was some 2 kb distant. The insertions showed biased inheritance following mitochondrial genome recombination. Recombination between H and J genomes was detected using the mitochondrial gene mutations acu-10, which causes a cytochrome oxidase defect, and cap-1, which confers chloramphenicol resistance. Fourteen of fifteen independently derived recombinants for these two genes were shown to have both DNA insertions. In a second series of H x J crosses, intragenic recombination between different cap-1 alleles was detected. These mutations are assumed to be in the large ribosomal RNA gene some 6 kb distant from the nearest insertion site. Each of eight independently derived cap-1 ⁺ recombinants had both DNA insertions. Despite their similar size and similar behaviour following recombination the insertions do not share extensive sequence homology.     

Isolation and identification of the Aspergillus nidulans gdhA gene encoding NADP-linked glutamate dehydrogenase

Summary The Neurospora crassa am gene was used as a heterologous probe to identify clones from two independently constructed Aspergillus nidulans gene libraries. These clones have a common HindIII 1.85 kb fragment. This A. nidulans nucleotide stretch hybridises to a N. crassa 2.7 kb BamHI fragment of wild type DNA but not to a co-migrating fragment from the DNA of the N. crassa am132 deletion mutant. One A. nidulans clone was shown to complement the N. crasse am132 deletion strain. The N. crassa transformants show low levels (5%) of heterologous glutamate dehydrogenase activity. The A. nidulans gdhA gene was found to locate in N. crassa at both the homologous (i.e. am) site as well as non-nomologous sites. Partial nucleotide analysis of the fragment has revealed the 5 end of the locus and considerable homology with the N. crassa am gene. We concluded that we have cloned the A. nidulans gdhA gene.Abbreviations bp base pairs - kb 1,000 bp - GDH NADP-Iinked glutamate dehydrogenase - NADP nicotinamide adenine dinuc leotide phosphate - SDS sodium dodecyl sulfate - phage lambda - DTT dithiothreitol - SSC 0.15 M NaCl, 0.015 Na₃ citrate pH 7.0     

Inversions and recombinations in mitochondrial DNA of the (SG-1) cytoplasmic mutant in two Neurospora species

Summary The mitochondrial DNAs of [SG-1] cytoplasmically-mutant and wild-type strains of Neurospora crassa and Neurospora sitophila were examined by comparative restriction endonuclease analyses. The mtDNA of N. sitophila wild type of Whitehouse differs from type II mtDNA of N. crassa by insertions of 3.3 kb in EcoRI-9, and 1.2 kb in EcoRI-3, and a deletion of 1.1 kb in EcoRI-5. These DNA heteromorphisms provided convenient markers for tracing N. crassa [SG-1] mtDNA during and after its transfer into N. sitophila. The [SG-1] cytoplasmic mutant in both N. crassa and N. sitophila has a distinctive inversion that connects the fragment EcoRI-4 with HindIII-10a. The [SG-1] mtDNA from N. crassa remained essentially intact after it was transferred by crosses into N. sitophila. In each species, a unique second inversion occured in the [SG-1] mtDNA after the transfer was made. In N. sitophila, polar recombination in heteroplasmons between [SG-1] and wild-type preferentially yields strains with mtDNAs that contain the maximum possible number of insertions in the cob and co-1 loci of the EcoRI-3 region of the mitochondrial chromosome.     

Nucleotide sequence of the Aspergillus niger trpC gene: structural relationship with analogous genes of other organisms

Summary The nucleotide sequence of the Aspergillus niger tryptophan C (trpC) gene was determined. Northern hybridization and S1-mapping experiments showed the presence of a 2.6 kb trpC poly(A)⁺ RNA with two very short (5 and 6 nucleotides) noncoding 5-regions. Comparison of the predicted amino acid sequence with that of trp gene proteins of pro- and eukaryotic organisms revealed three functional domains (G, C, F) in the A. niger TrpC protein which catalyse the glutarnine amidotransferase reaction (GAT), the indole-3-glycerol phosphate synthase reaction (IGPS) and the N-(5-phosphoribosyl) anthranilate isomerase reaction (PRAI), respectively. These domains are highly conserved and bordered by short areas showing less homology. Within the F domain of the trpC gene in A. niger, A. nidulans and Neurospora crassa, a region encoding 30 amino acids was found which is absent in the analogous genes of Saccharomyces cerevisiae and prokaryotic organisms. This region has features of a mutated in-phase intron.     

Mitochondrial genetics of Coprinus: Recombination of mitochondrial genomes

Summary The formation of the sexual mycelium or dikaryon in the basidiomycete Coprinus cinereus involves exchange and migration of nuclei without accompanying exchange of mitochondria. The dikaryotic growth which appears around the periphery of mated monokaryons has exclusively the mitochondrial genome of the recipient cells. Recombination of mitochondrial genomes is not, however, precluded during dikaryosis. Using monokaryons with different mitochondrial gene mutations, [acu-10] causing cytochrome aa ₃ deficiency and[cap-1.1] conferring resistance to chloramphenicol, it was shown that recombinant mitochondria arise in the zone of contact of mated monokaryons.     

Organization and nucleotide sequence of ribosomal RNA genes on a circular 73 kbp DNA from the colourless flagellate Astasia longa

Summary Three tandemly arranged repeats (A, B, C) of 16S and 23S rDNA, and one supplementary (S) 16S rDNA adjacent to the 16S rDNA of repeat A, are present within an 18 kbp segment of a circular 73 kbp DNA from the colourless flagellate Astasia longa. The repeat units are separated by a short region containing a 5S rRNA gene and a gene for tRNA-Val (UAC). Sequence comparisons reveal 78%, 81%, and 67% identical nucleotides of the 23S rDNA (A), the 16S rDNA (B), and the 5S rDNA (A), respectively, with the corresponding genes of the Euglena gracilis chloroplast genome. As in Euglena chloroplasts, the 3-terminal protion of the 23S rDNA is homologous to the 4.5S rRNA gene of higher plant chloroplast genomes. These results are supportive of a common evolutionary origin for the Astasia 73 kbp DNA and the Euglena 145 kbp chloroplast DNA.     

The mitochondrial genome of the aquatic phycomycete Allomyces macrogynus. Physical mapping and mitochondrial DNA instability

Summary A physical map of the mitochondrial genome of the aquatic phycomycete Allomyces macrogynus strain Burma 3–35 (35°C) has previously been published (Borkhardt and Delius 1983). This map has been extended in this study by locating 37 additional recognition sites for five new restriction enzymes in the mitochondrial genome. Homologous regions for the genes coding for cytochrome oxidase subunits 1, 2, and 3, apocytochrome b, ATPase subunits 6 and 9, the small and large ribosomal RNA, URF1, URF5, and perhaps urfa, a presumptive gene hitherto found only in the mitochondrial genome of the fission yeast Schizosaccharomyces pombe, were located in the mitochondrial genome of A. macrogynus by heterologous hybridizations with specific, mitochondria) gene probes from Saccharomyces cerevisiae, Aspergillus nidulans, Neurospora crassa, and S. pombe. The mitochondrial gene order in A. macrogynus was found to be identical to that of A. arbuscula; a gene order hitherto found only among members of the family Blastocladiaceae. Spontaneous insertion mutations have been found to occur quite frequently in the mitochondrial genome of A. macrogynus. In all mutated mitochondrial genomes so far studied, insertions have been located in a specific region located between the genes coding for the ATPase subunit 9 and the large ribosomal RNA. In two of the mutated mitochondrial genomes the insertional event(s) resulted in the presence of mitochondrial DNA molecules differing in size by multiples of approximately 70 base pairs.     

Mapping of rRNA genes by integration of hybrid plasmids in Schizosaccharomyces pombe

Summary The major rRNA genes of the fission yeast Schizosaccharomyces pombe were mapped on chromosome III by plasmid integration. The integration vector YIp33 containing S. cerevisiae LEU2 gene was combined with the S. pombe rDNA. Since LEU2 complements S. pombe leu1 deficiency, it could be used as the genetic marker for integration. The 10.4 kb rDNA repeat contained ARS sequence, and therefore 2.4 kb and 0.7 kb subfragments not containing ARS were subcloned into YIp33 and transformed leu1 S. pombe cells to Leu⁺. Genetic analyses of the transformants indicated that the integrated rDNA resides in the long arm of the shortest chromosome III, tightly linked to ade5 (1.4 cM). This result is consistent with our previous finding that the DAPI-stained smallest chromosomes were associated with the nucleolus (Umesono et al. 1983).Abbreviations ARS autonomously replicating sequence - DAPI 4,6-diamidino-2-phenylindole - kb kilo base pairs - rDNA DNA segment containing ribosomal RNA genes - rRNA ribosomal RNA     

The chloroplast trnP-trnW-petG gene cluster in the mitochondrial genomes of Beta vulgaris,B. trigyna and B. webbiana: evolutionary aspects

The chloroplast trnP-trnW-petG gene cluster has been identified in the mitochondrial DNA (mtDNA) of sugar beet (Beta vulgaris). The chloroplast-derived trnW gene is transcribed in the mitochondria; the other two genes, however, do not seem to be transcribed. This gene cluster is also present in the mitochondrial genomes of two wild Beta species, B. trigyna and B. webbiana. Sugar beet and the two wild relatives share 100% sequence identity in the coding regions of both the mitochondrial trnP and trnW genes. On the other hand, the petG genes from the wild Beta mtDNAs were found to be disrupted either by a 5-bp duplication (B. trigyna) or by a deletion of the 5 region (B. webbiana). A data-base search revealed that a conserved sequence of 60 bp is present in the trnP-trnW intergenic region of the mitochondrial genomes of the three Beta species as well as in other higher plants, including wheat and maize, and that the conserved sequence is absent from the chloroplast counterpart. Our results thus favour the hypothesis of a monophyletic origin of the trnP-trnW-petG cluster found in the plant mitochondrial genomes examined.     

The chloroplast genome of Euglena gracilis: the mosaic structure of a DNA segment linking the extra 16S rRNA gene with the rrn operon A

Summary We have completed the analysis of a DNA segment of the chloroplast genome of Euglena gracilis Klebs, Z-strain, which links the 3 end of the extra 16S rRNA gene with the 5 end of the 16S rRNA gene of the rrn operon A. This region is a mosaic of several structural elements and contains an intact rrn interoperon spacer of 1,080 bp, an extra 5S rRNA gene, an open reading frame for 406 codons (ORF 406) which is flanked by short inverted repeats and a short direct repeat originating from the rrn interoperon spacer. It seems that a once complete rrn operon underwent in the past an insertion/deletion event leaving intact the 16S and 5S rRNA but totally excising the 16S-23S intergenic spacer and the 23S rRNA gene. Instead a protein coding gene of yet unknown function was inserted along with other structural elements.     

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.     

A DNA sequence in Saccharomyces exiguus is homologous with the HO gene of Saccharomyces cerevisiae

Summary The DNA of Saccharomyces exiguus was analyzed by Southern hybridization using cloned MATa, MAT, and HO genes of Saccharomyces cerevisiae as probes. It was shown that S. exiguus has a DNA sequence homologous with the HO gene of S. cerevisiae and that this DNA sequence is on a chromosome of about 940 kb of DNA in S. exiguus. However, there is no DNA sequence in S. exiguus that is homologous with the MAT genes of S. cerevisiae.     

A new senescence-inducing mitochondrial linear plasmid in field-isolated Neurospora crassa strains from India

Summary Several field-collected strains of Neurospora crassa from the vicinity or Aarey, Bombay, India, are prone to precocious senescence and death. Analysis of one strain, Aarely-1e, demonstrated that the genetic determinants for the predisposition to senescence are maternally inherited. The senescence-prone strains contain a 7-kb, linear, mitochondrial DNA plasmid, maranhar, which is not present in long-lived isolates from the same geographical location. The maranhar plasmid has inverted terminal repeats with protein covalently bound at the 5 termini. Molecular hybridization experiments have demonstrated no substantial DNA sequence homology between the plasmid and the normal mitochondrial (mtDNA) and nuclear genomes of long-lived strains of N. crassa. Integrated maranhar sequences were detected in the mtDNAs of two cultures derived from Aarey-1e, and mtDNAs with the insertion sequences accumulated during subculturing. Nucleotide sequence analysis of cloned fragments of the two insertion sequences demonstrates that that they are flanked by long inverted repeats of mtDNA. The senescence syndrome of the maranhar strains, and the mode of integration of the plasmid, are reminiscent of those seen in the kalilo strains of N. intermedia. Nonetheless, there is no detectable nucleotide sequence homology between the maranhar and kalilo plasmids.     

Extrachromosomal genetics of Claviceps purpurea

Summary Claviceps purpurea strain K1 contains several linear mitochondrial plasmids comparable to those of higher plants (Tudzynski et al. 1983). Screening of ten Claviceps wild strains from different geographic origin revealed the presence of similar plasmids in at least five strains, indicating a widespread occurrence of these genetic traits. Whereas in strain K1 plasmids have no homology to high-molecular-weight mtDNA, in two of the other wild strains (W3 and W7) sequences homologous to plasmids of strain K1 are integrated in the mt genomic DNA. Physical and genetic maps of strains W3, W7 and K1 were established. Despite completely different physical maps, the relative orientation of 6 mt genes is identical. A DNA sequence homologous to plasmids of strain K1 (termed ) was found to be integrated in both W3 and W7 at the same site: at the 5-part of the 1rRNA gene. A second region () was localized near the srRNA gene of W7 only. These findings are discussed with respect to possible transposon-like properties of the Claviceps purpurea plasmids.     

Molecular analysis of mitochondrial DNA and its inheritance in Epilobium

Summary The mitochondrial genome of four Epilobium species has been characterized by restriction analysis and hybridizations with gene probes from Oenothera. Mitochondrial DNA of Epilobium has a complex restriction fragment pattern and an estimated size of about 320 kb. All species exhibit specific restriction patterns. Plasmid-like DNA molecules of 0.3 kb to 1.2 kb are found in preparations of undigested nucleic acids of mitochondria from E. montanum, E. watsonii, and E. lanceolatum. In contrast, the mitochondria of E. hirsutum contain double-stranded RNAs of 2.7 kb. The location of the genes for cytochrome c oxidase subunits I and III on the mitochondrial DNA seems to be conserved in those species analyzed. However, the genes for subunit II of this complex, and for the alpha subunit of ATPase, are located on different restriction fragments in the mitochondrial genomes of certain species. The location of the COX II gene on different BamHI fragments in E. watsonii and E. lanceolatum has been used for the analysis of mitochondrial inheritance in reciprocal hybrids. Like the plastids, mitochondria are inherited maternally in Epilobium.Abbreviations kb kilobase pairs - mtDNA mitochondrial DNA     

Cryptic DNA plasmids of the heterothallic yeast Saccharomycopsis crataegensis

Summary Three DNA plasmids, designated pScrl-1, pScrl-2, and pScrl-3 have been found in a strain of the heterothallic yeast Saccharomycopsis crataegensis (NRRL Y-5902). pScrl-l, -2 and -3 are, respectively, 15, 7, and 5 kilobase pairs (kbp) in size. Based on the results of exonuclease digestions, all three plasmids appear to be linear molecules with blocked 5 ends. All three plasmids also have a lower buoyant density than does nuclear DNA of S. crataegensis. The two lower molecular weight plasmids hybridize strongly with one another, but only weakly to the higher molecular weight plasmid. Two of four related S. crataegensis strains surveyed were found to contain two plasmids that are of the same size as the two larger plasmids of Y-5902. Evidence is presented indicating that the plasmids in strain Y-5902 reside in the cytosol since they were found not to be located within the major organelles (mitochondria and nuclei).