A group-I intron in the mitochondrial small subunit ribosomal RNA gene of Sclerotinia sclerotiorum

A 1 380-bp intervening sequence within the mitochondrial small subunit ribosomal RNA (mt SSU rRNA) gene of the fungus Sclerotinia sclerotiorum has been sequenced and identified as a group-I intron. This is the first report of an intron in the mt SSU rRNA gene. The intron shows close similarity in secondary structure to the subgroup-IC2 introns from Podospora (ND3i1, ND5i2, and COIi5) and Neurospora (ND5i1). The intron has an open reading frame (ORF) that encodes a putative protein of 420 amino acids which contains two copies of the LAGLI-DADG motif. The ORF belongs to a family of ORFs identified in Podospora (ND3i1, ND4Li1, ND4Li2, ND5i2, and COIi5) and Neurospora (ND5i1). The putative 420-aa polypeptide is also similar to a site-specific endonuclease in the chloroplast large subunit ribosomal RNA (LSU rRNA) gene of the green alga Chlamydomonas eugametos. In each clone of S. sclerotiorum examined, including several clones which were sampled over a 3-year period from geographically separated sites, all isolates either had the intron or lacked the intron within the mt SSU rRNA gene. Screening by means of Southern hybridization and PCR amplification detected the intron in the mt SSU rRNA genes of S. minor, S. trifoliorum and Sclerotium cepivorum, but not in other members of the Sclerotiniaceae, such as Botrytis anamorphs of Botryotinia spp., or in other ascomycetous and basidiomycetous fungi.     

Complex group-I introns in nuclear SSU rDNA of red and green algae: evidence of homing-endonuclease pseudogenes in the Bangiophyceae

 The green alga Scenedesmus pupukensis and the red alga Porphyra spiralis contain large group-IC1 introns in their nuclear small subunit ribosomal RNA genes due to the presence of open reading frames at the 5′ end of the introns. The putative 555 amino-acid Scenedesmus-encoded protein harbors a sequence motif resembling the bacterial S9 ribosomal proteins. The Porphyra intron self-splices in vitro, and generates both ligated exons and a full-length intron RNA circle. The Porphyra intron has an unusual structural organization by encoding a potential 149 amino-acid homing-endonuclease-like protein on the complementary strand. A comparison between related group-I introns in the Bangiophyceae revealed homing-endonuclease-like pseudogenes due to frame-shifts and deletions in Porphyra and Bangia. The Scenedesmus and Porphyra introns provide new insights into the evolution and possible novel functions of nuclear group-I intron proteins. Received: 26 May / 25 August 1999     

The cyanobacterial origin and vertical transmission of the plastid tRNALeu group-I intron

We have surveyed the distribution and reconstructed the phylogeny of the group-I intron that is positioned in the anticodon loop of the tRNA^Leu gene in cyanobacteria and several plastid genomes. Southern-blot and PCR analyses showed that the tRNA^Leu intron is found in all 330 land plants that were examined. The intron was also found, and sequenced, in all but one of nine charophycean algae examined. Conversely, PCR analyses showed that the tRNA^Leu group-I intron is absent from the red, cryptophyte and haptophyte algae, although it is present in three members of the heterokont lineage. Phylogenetic analyses of the intron indicate that it was present in the cyanobacterial ancestor of the three primary plastid lineages, the Rhodophyta, Chlorophyta, and Glaucocystophyta. Its present-day distribution in plastids is consistent with a history of strictly vertical transmission, with no losses in land plants, several losses among green algae, and nearly pervasive loss in the Rhodophyta and its secondary derivatives. Received: 1 August / 22 September 1999     

Identification of group-I introns in the 28s rDNA of the entomopathogenic fungus Beauveria brongniartii

The length of the 28s ribosomal DNA differs significantly between two strains (Bt102 and Bt114) of the entomopathogenic fungus Beauveria brongniartii. RFLP analysis on PCR products revealed the presence of three insertional elements of 350–450 bp in strain Bt114. One of the insertions has been cloned and sequenced and shown to possess all the characteristic sequences and secondary structures of a group-IC intron. Its length is 428 bp and it is devoid of any long open reading frame. The distribution of this intron elsewhere in the genome of Bt114, as well as in the chromosomal ribosomal DNA, was studied. It seems to be present as seven copies in different genes not corresponding to the mitochondrial DNA. The presence of the intron in other strains of B. brongniartii was examined by the hybridization method. Some of them seemed to possess introns with a similar core although others presented no homology with the cloned fragment.     

Primary and secondary structure analyses of the rDNA group-I introns of the Zygnematales (Charophyta)

The Zygnematales (Charophyta) contain a group-I intron (subgroup ICl) within their nuclear-encoded small subunit ribosomal DNA (SSU rDNA) coding region. This intron, which is inserted after position 1506 (relative to the SSU rDNA ofEscherichia coli), is proposed to have been vertically inherited since the origin of the Zygnematales approximately 350–400 million years ago. Primary and secondary structure analyses were carried out to model group-I intron evolution in the Zygnematales. Secondary structure analyses support genetic data regarding sequence conservation within regions known to be functionally important for in vitro self-splicing of group-I introns. Comparisons of zygnematalean group-I intron secondary structures also provided some new insights into sequences that may have important roles in in vivo RNA splicing. Sequence analyses showed that sequence divergence rates and the nucleotide compositions of introns and coding regions within any one taxon varied widely, suggesting that the 1506 group-I introns and rDNA coding regions in the Zygnematales evolve independently.     

Amplification of the yeast nuclear gene MRS3 confers suppression of a mitochondrial RNA splice defect

Summary The MRS3 gene cloned in the multicopy plasmid YEp13 suppresses the mitochondrial splice defect exerted by mutation M1301 in the group II intron bIl. In this article we report on the behavior of the MRS3 gene cloned in the integration vector pEMBLYi27 and in the CEN4-ARS vector YCp50. Transformation of mutant M1301 cells with these recombinant vectors produced transformants, the majority of which showed the original splice defect and contained the recombinant vectors in single or low copy; a minority, however, was splicing competent and showed exceptionally high copy numbers of the MRS3 gene. These latter transformants had either the pEMBLYi27/MRS3 sequence repeated at least 20 times in tandem at the chromosomal site of the MRS3 gene or they had the YCp50/MRS3 sequence established as a multicopy plasmid lacking the copy number control usually exerted by the CEN4 sequence in this plasmid.     

Homologous maturase-like proteins are encoded within the group I introns in different mitochondrial genes specifying Yarrowia lipolytica cytochrome c oxidase subunit 3 and Saccharomyces cerevisiae apocytochrome b

A mitochondrial cox3 gene in the alkane yeast, Yarrowia lipolytica, encodes a subunit-3 protein of cytochrome c oxidase, and contains a 1044 base-pair-long intron, as compared with the corresponding intronless gene in Saccharomyces cerevisiae. The intron belongs to a group I intron as determined by the cDNA sequence for the splicing sites as well as the predicted RNA secondary structure. Remarkably, this intron could code for a protein of 206 amino-acid residues which showed 63% similarity with an RNA maturase encoded by the second intron of the mitochondrial apocytochrome b gene in S. cerevisiae. Both introns occurred within the conserved exon sequence, 5-TT(G/C)AGGTGC-3, suggesting the possible transposition of a common ancestral intron.     

Assembly of the mitochondrial membrane system. Organization of yeast mitochondrial DNA in the Oli1 region

Summary The mitochondrial DNA (mtDNA) of a cytoplasmic petite mutant (DS401) of Saccharomyces cerevisiae genetically marked for the ATPase proteolipid, serine tRNA and varl genes has been characterized by restriction endonuclease analysis and DNA sequencing. The DS401 mtDNA segment is 5.3 kb long spanning the region between 79.1 and 86.8 units of the wild type genome. Most of the DS401 mtDNA consists of A+T rich sequences. In addition, however, there are ten short sequences with a high content of G+C and two sequences that have been identified as the ATPase proteolipid and the serine tRNA genes. The two genes map at 81 and 83 units and are transcribed from the same DNA strand. Even though there are other possible coding sequences in the DNA segment, none are sufficiently long to code for a gene product of the size of the varl protein. Based on the relative organization of the G+C rich clusters and genes, a model has been proposed for the processing of mitochondria) RNA. This model postulates the existence of mitochondrial double strand specific RNases that cleave the RNA at the G+C clusters.     

A long open reading frame in the mitochondrial LSU rRNA group-I intron of Cryphonectria parasitica encodes a putative S5 ribosomal protein fused to a maturase

A 4238-bp intervening sequence within the highly conserved U11 region of the mitochondrial large subunit ribosomal RNA gene of the fungus Cryphonectria parasitica Ep155 has been sequenced and identified to be a group-I intron. This is the largest group-I intron reported to-date for fungal mitochondrial genomes. The intron contains an 851-codon open reading frame encoding a putative, but complete, small-subunit ribosomal protein of 510 amino acids which is fused at its carboxyl terminus to a 311 amino-acid polypeptide representing a typical maturase-like protein. A short open reading frame of 83 amino acids with some similarity to maturases, but lacking a translation-initiation codon, was also noted at the 3′ end of the intron. The unusual size of the intron and the arrangement of the open and truncated reading frames suggest that this segment of the mtDNA of C. parasitica has arisen by a fusion of components from two or more different introns, possibly involving the re-location of intronic genes. Received: 7 August / 15 December 1998     

Cloning and characterization of MRP10, a yeast gene coding for a mitochondrial ribosomal protein

The nuclear gene MRP10 of Saccharomyces cerevisiae was cloned by complementation of a respiratory deficient mutant N518/L1. This mutant is defective in mitochondrial translation and shows a tendency to accumulate deletions in mitochondrial DNA (ρ ^–). Analysis revealed Mrp10p to be a component of the 37 S subunit of the mitochondrial ribosomes. Disruption of MRP10 in a haploid strain of yeast elicits a phenotype identical to that of the original mutant. The respiratory defect of the null mutant is rescued by re-introducing the MRP10 gene in a wild-type mitochondrial DNA background. These results indicate that Mrp10p belongs to the class of yeast mitochondrial ribosomal proteins that are essential for translation. Searches of current databases failed to reveal any homologs among known bacterial or eucaryotic cytoplasmic ribosomal proteins. Some sequence similarity, however, was detected between Mrp10p and Yml37p, previously identified as a component of the yeast mitochondrial 50 S ribosomal subunit. Received: 21 November 1996     

Group-I introns in the cytochrome c oxidase genes of Dictyostelium discoideum : two related ORFs in one loop of a group-I intron, a cox1/2 hybrid gene and an unusually large cox3 gene

 The DNA sequences of cytochrome oxidase (subunits 1, 2 and 3) genes of the cellular slime mold Dictyostelium discoideum mitochondria were determined. The genes for subunits 1 and 2 have a single continuous ORF (COX1/2) which contains four group-I introns. The insertion sites of the two group-I introns (DdOX1/2.2 and DdOX1/2.3) coincide with those of fungal and algal group-I introns, as well as a liverwort group-I intron, in the cytochrome oxidase subunit 1. Interestingly, intron DdOX1/2.2 has two free-standing ORFs in a loop (L8) which have similar amino-acid sequences and are homologous to ai4 DNA endonuclease (I-Sce II) and bi4 RNA maturase found in group-I introns of Saccharomyces cerevisiae mitochondrial DNA. Two group-I introns (DdOX1/2.3 and DdOX1/2.4) also have a free-standing ORF in loop 1 and loop 2, respectively. These results show that these group-I introns and the intronic ORFs have evolved from the same ancestral origin, but that these ORFs have been propagated independently. Received: 1 May / 16 September 1996     

Two group I mitochondrial introns in the cob-box and coxI genes require the same MRS1/PET157 nuclear gene product for splicing

Summary We have studied the role of the product of the nuclear gene PET157 in mitochondrial pre-mRNA splicing. Cytoduction experiments show that a mitochondrial genome deleted for the three introns bI3, aI5 and aI6 is able to suppress the pet157-1 mutation: the strain recovers respiratory competency indicating that the product of the PET157 gene is only required for mitochondrial premRNA splicing. Characterization of the high molecular weight pre-mRNAs which accumulate in the pet157 mutant demonstrate that the product of the PET157 gene is required for the excision of two group I introns bI3 and aI6 (corresponding to aI5) located in the cob-box and coxI genes respectively. Furthermore, the pet157 mutant strain accumulates the bI3 maturase in the form of a polypeptide of 50K (p50) previously observed in mitochondrial mutants defective in the excision of bI3. We have shown by restriction analysis and allelism tests that the pet157-1 mutation is allelic to the nuclear mrs1 mutation, previously described as specifically blocking the excision of bI3. Finally, revertants obtained by the deletion of bI3 or aI6 from the mitochondrial DNA were isolated from the MRS1 disrupted allele, confirming the involvment of the product of the MRS1/PET157 gene in the excision of the two introns bI3 and aI6.     

Large size and complex structure of mitochondrial DNA in two nonflowering land plants

Summary We report the first estimates of genome size and complexity for mitochondrial DNAs (mtDNAs) from nonflowering land plants. The mtDNA of Onoclea sensibilis (sensitive fern) is approximately 300 kb in size, while that of Equisetum arvense (common horsetail) is at least 200 kb. Sufficient mtDNA of Onoclea was available to permit an estimation of the copy number and a linkage analysis of nine mitochondrial genes. Six of these genes appear to be present in only one or two copies in the Onoclea genome, whereas three other genes are present in multiple copies. Five of the approximately ten genes encoding 26S rRNA are located on a large, >10kb, dispersed repeat that also contains closely linked genes for 18S rRNA and the alpha subunit of ATPase (atpA). The other 26S genes belong to a second dispersed repeat family of >8 kb whose elements do not contain any other identified genes. Because flowering plant mtDNAs are also large and contain dispersed, gene-containing, repeats, it appears that these features arose early in the evolution of land plants, or perhaps even in their green algal ancestors.     

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     

Ts mutations in mitochondrial tRNA genes: characterization and effects of two point mutations in the mitochondrial gene for tRNAphe in Saccharomyces cerevisiae

Two new mitochondrial mutations conferring heat sensitivity on glycerol medium to the cells that carry them and affecting mitochondrial protein synthesis were investigated. Both map in the mitochondrial tRNAphe gene and have C-to-U transitions, one at position 2 (ts22b16) and the other at 62 (ts1345). The latter mutation clearly affects the 3′ end-maturation of tRNAphe, while the former presents normal patterns of both tRNA processing and amino-acylation. The defective phenotype resulting from the ts22b16 mutation can be corrected by over-expressing either the mitochondrial elongation factor EF-Tu or the mutated form of the tRNA. These results suggest that this mutation's primary effect might involve modified interactions during the ternary complex formation. Received: 27 July / 14 October 1997     

Synthesis and function of the mitochondrial intron —encoded bI4 RNA maturase from Saccharomyces cerevisiae

Summary We have analyzed the expression and function of the intron-encoded bI4 maturase when frame-shift mutations in the upstream exon alter the translational process. By constructing secondary cis-acting mutations within the b14 intron, we observed (1) that the bI4 maturase is still translated in the presence of the upstream mutation, albeit in very low amounts, and (2) that the limited amounts of bI4 maturase made under these conditions is no longer able to promote the splicing process of the aI4 intron. These observations, which further strengthen the maturase model, strongly suggest that bI4 maturase acts sequentially on the bI4 intron and then on the aI4 intron.     

Physical and gene organization of mitochondrial DNA from the fertile cytoplasm of sugarbeet (Beta vulgaris L.)

We have constructed a complete physical map of the mitochondrial genome from the male-fertile cytoplasm of sugarbeet. The entire sequence complexity can be represented on a single circular master chromosome of 358 kb. This master chromosome contains three copies of one recombinationally active repeat sequence, with two copies in direct orientation and the other in inverted orientation. The positions of the rRNA genes and of 23 polypeptide genes, determined by filter hybridization, are scattered throughout the genome, with triplicate rrn26 genes located partially or entirely within the recombination-repeat elements. Three ribosomal-protein genes (rps1A, rps14 and rps19) were found to be absent from sugarbeet mtDNA. Our results also reveal that at least six regions homologous with cpDNA are dispersed in the mitochondrial genome.