Mitochondrial DNA sequence analysis of the cytochrome oxidase subunit II gene from Podospora anserina. A group IA intron with a putative alternative splice site

A 5 kb region of the 95 kb mitochondrial genome of Podospora anserina race s has been mapped and sequenced (1 kb = 10(3) base-pairs). This DNA region is continuous with the sequence for the ND4L and ND5 gene complex in the accompanying paper. We show that this sequence contains the gene for cytochrome oxidase subunit II (COII). This gene is 4 kb in length and is interrupted by a subgroup IB intron (1267 base-pairs (bp) in length) and a subgroup IA intron (1992 bp in length). This group IA intron has a long open reading frame (ORF; 472 amino acid residues) discontinuous with the upstream exon sequence. A putative alternative splice site is present, which brings the ORF into phase with the 5' exon sequence. The 5'- and 3'-flanking regions of the COII gene contain G + C-rich palindromic sequences that resemble similar sequences flanking many Neurospora crassa mitochondrial genes.     

Protein encoded by the third intron of cytochrome b gene in Saccharomyces cerevisiae is an mRNA maturase. Analysis of mitochondrial mutants, RNA transcripts proteins and evolutionary relationships

We have established the nucleotide sequence of the wild-type and that of a trans-acting mutant located in the third (bi3) intron of the Saccharomyces cerevisiae mitochondrial cytochrome b gene. The intron, 1691 base-pairs long, has an open reading frame 1045 base-pairs long, in phase with the preceding exon and the mutation replaces the evolutionarily conserved Gly codon of the second consensus motif by an Asp codon and blocks the formation of mature cytochrome b mRNA. Splicing intermediates of 5300 and 3900 bases with unexcised bi3 intron and a characteristic novel polypeptide (p50), the size of which corresponds to the chimeric protein encoded by upstream exons and the bi3 intronic open reading frame (ORF), accumulate in this and other bi3 splicing-deficient mutants. We conclude that the protein encoded by the bi3 ORF is a specific mRNA maturase involved in the splicing of the cytochrome b mRNA. The open reading frame of the third intron is remarkably similar to that of the unique intron of the cytochrome b gene (cob A) of Aspergillus nidulans. Both are located in exactly the same position and possibly derive from a recent common ancestor by a horizontal transfer. We have established the nucleotide sequence of an exonic mutant located in the B3 exon. This missense mutation changes the Phe codon 151 into a Cys codon and leads to the absence of functional cytochrome b but does not affect splicing. Finally, we have studied the splicing pathway leading to the synthesis of cytochrome b mRNA by analysing, in a comprehensive manner, the 22 splicing intermediates of several mutants located in bi3.     

The mitochondrial genome of the fission yeast Schizosaccharomyces pombe. The cytochrome b gene has an intron closely related to the first two introns in the Saccharomyces cerevisiae cox1 gene

The DNA sequence of the cob region of the Schizosaccharomyces pombe mitochondrial DNA has been determined. The cytochrome b structural gene is interrupted by an intron of 2526 base-pairs, which has an open reading frame of 2421 base-pairs in phase with the upstream exon. The position of the intron differs from those found in the cob genes of Saccharomyces cerevisiae, Aspergillus nidulans or Neurospora crassa. The Sch. pombe cob intron has the potential of assuming an RNA secondary structure almost identical to that proposed for the first two cox1 introns (group II) in S. cerevisiae and the p1-cox1 intron in Podospora anserina. It has most of the consensus nucleotides in the central core structure described for this group of introns and its comparison with other group II introns allows the identification of an additional conserved nucleotide stretch. A comparison of the predicted protein sequences of group II intronic coding regions reveals three highly conserved blocks showing pairwise amino acid identities of 34 to 53%. These regions comprise over 50% of the coding length of the intron but do not include the 5' region, which has strong secondary structural features. In addition to the potential intron folding, long helical structures involving repetitive sequences can be formed in the flanking cob exon regions. A comparison of the Sch. pombe cytochrome b sequence with those available from other organisms indicates that Sch. pombe is evolutionarily distant from both budding yeasts and filamentous fungi. As was seen for the Sch. pombe cox1 gene (Lang, 1984), the cob exons are translated using the universal genetic code and this distinguishes Sch. pombe mitochondria from all other fungal and animal mitochondrial systems.     

Nucleotide sequence and intron structure of the apocytochrome b gene of Neurospora crassa mitochondria

The sequence of the apocytochrome b (cob) gene of Neurospora crassa has been determined. The structural gene is interrupted by two intervening sequences of approximately 1260 bp each. The polypeptide encoded by the exons shows extensive homology with the cob proteins of Aspergillus nidulans and Saccharomyces cerevisiae (79% and 60%, respectively). The two introns are, however, located at sites different from those of introns in the cob genes of A. nidulans and S. cerevisiae (which contain highly homologous introns at the same site within the gene). The introns share several short regions of sequence homology (10-12 bp long) with each other and with other fungal mitochondrial introns. Moreover, the second intron contains a 50 nucleotide long sequence that is highly homologous with sequences within every ribosomal intron of fungal mitochondria sequenced to date. The conserved sequences may allow the formation of a core secondary structure, which is nearly identical in many mitochondrial introns. The conserved secondary structure may be required for intron splicing. The second intron contains an open reading frame, continuous with the preceding exon, of approximately 290 codons. Two stretches of 10 amino acid residues, conserved in many introns, are present in the open reading frame.     

The self-splicing intron in the Neurospora apocytochrome b gene contains a long reading frame in frame with the upstream exon.

We have determined the DNA sequence of intron 1 and flanking exons in the mitochondrial apocytochrome b gene of the Neurospora laboratory strain 74A and the natural isolate North Africa. In contrast to a previous report, we find that this intron contains an open reading frame (ORF) of 951 bases in frame with the upstream exon. The putative intron-encoded protein resembles those of other intron ORFs with respect to length, calculated isoelectric point, and proportion of basic, acidic, polar, and non-polar amino acids; however, no amino acid sequences resembling the "decapeptides" characteristic of maturase-like ORFs were found. Coupled with the previous finding that this intron is capable of self-splicing in vitro in the absence of proteins, the observations discussed here raise the possibility that other introns with long, in-frame ORFs may also be capable of RNA-catalyzed splicing in vitro.     

Three class I introns in the ND4L/ND5 transcriptional unit of Neurospora crassa mitochondria

The overlapping ND4L and ND5 genes of Neurospora crassa mitochondria are interrupted by one and two intervening sequences, respectively, of about 1,490, 1,408 and 1,135 bp in length. All three intervening sequences are class I introns and as such have the potential to fold into the conserved secondary structure that has been proposed for the majority of fungal mitochondrial introns. They contain long open reading frames (ORFs; from 306 to 425 codons long) that are continuous and in frame with the upstream exon sequences. These ORFs contain the conserved decapeptide-encoding sequences that are characteristic of the ORFs present in most class I introns. Extensive homology exists among the ORFs encoded by the ND4L intron, ND5 intron 1, and the second intron of the N. crassa oli2 gene. Also, internal repeats of about 130 amino acid residues are present twice in each of these three ORFs, suggesting that a duplication event may have occurred in the formation of these ORFs. The ND4L intron shares extensive homology (at the levels of both primary and proposed secondary structures) with the self-splicing intervening sequence present in the Tetrahymena nuclear rRNA gene. This homology includes but is not limited to the core secondary structure, as peripheral structural elements are also conserved in the two introns.     

Incipient mitochondrial evolution in yeasts. II. The complete sequence of the gene coding for cytochrome b in Saccharomyces douglasii reveals the presence of both new and conserved introns and discloses major differences in the fixation of mutations in evolution.

We have determined the complete sequence of the mitochondrial gene coding for cytochrome b in Saccharomyces douglasii. The gene is 6310 base-pairs long and is interrupted by four introns. The first one (1311 base-pairs) belongs to the group ID of secondary structure, contains a fragment open reading frame with a characteristic GIY ... YIG motif, is absent from Saccharomyces cerevisiae and is inserted in the same site in which introns 1 and 2 are inserted in Neurospora crassa and Podospora anserina, respectively. The next three S. douglasii introns are homologous to the first three introns of S. cerevisiae, are inserted at the same positions and display various degrees of similarity ranging from an almost complete identity (intron 2 and 4) to a moderate one (intron 3). We have compared secondary structures of intron RNAs, and nucleotide and amino acid sequences of cytochrome b exons and intron open reading frames in the two Saccharomyces species. The rules that govern fixation of mutations in exon and intron open reading frames are different: the relative proportion of mutations occurring in synonymous codons is low in some introns and high in exons. The overall frequency of mutations in cytochrome b exons is much smaller than in nuclear genes of yeasts, contrary to what has been found in vertebrates, where mitochondrial mutations are more frequent. The divergence of the cytochrome b gene is modular: various parts of the gene have changed with a different mode and tempo of evolution.     

Transposition of an intron in yeast mitochondria requires a protein encoded by that intron

The optional 1143 bp intron in the yeast mitochondrial 21S rRNA gene (omega +) is nearly quantitatively inserted in genetic crosses into 21S rRNA alleles that lack it (omega -). The intron contains an open reading frame that can encode a protein of 235 amino acids, but no function has been ascribed to this sequence. We previously found an in vivo double-strand break in omega - DNA at or close to the intron insertion site only in zygotes of omega + X omega - crosses that appears with the same kinetics as intron insertion. We now show that mutations in the intron open reading frame that would alter the translation product simultaneously inhibit nonreciprocal omega recombination and the in vivo double-strand break in omega - DNA. These results provide evidence that the open reading frame encodes a protein required for intron transposition and support the role of the double-strand break in the process.     

Splicing defective mutants of the COXI gene of yeast mitochondrial DNA: initial definition of the maturase domain of the group II intron aI2.

Six mutations blocking the function of a seven intron form of the mitochondrial gene encoding subunit I of cytochrome c oxidase (COXI) and mapping upstream of exon 3 were isolated and characterized. A cis-dominant mutant of the group IIA intron 1 defines a helical portion of the C1 substructure of domain 1 as essential for splicing. A trans-recessive mutant confirms that the intron 1 reading frame encodes a maturase function. A cis-dominant mutant in exon 2 was found to have no effect on the splicing of intron 1 or 2. A trans-recessive mutant, located in the group IIA intron 2, demonstrates for the first time that intron 2 encodes a maturase. A genetic dissection of the five missense mutations present in the intron 2 reading frame of that strain demonstrates that the maturase defect results from one or both of the missense mutations in a newly-recognized conserved sequence called domain X.     

Expression of the split gene cob in yeast: Evidence for a precursor of a “maturase” protein translated from intron 4 and preceding exons

Intron 4 (14) of the split gene cob in mitochondrial DNA contains a long open reading frame in phase with the preceding exon. Mutations in this intron block the excision of the 14 sequence from the cob precursor RNA and, at the same time, generate a series of new polypeptides, parts of which apparently result from translation of 14 sequences. We sequenced six mutations clustered in the upstream part of the open reading frame, about 340 bp from the exon-intron boundary (box9 cluster). Four are base pair exchanges in the same triplet of this region; these form the polypeptides typical for 14 plus a trans-acting product encoded by 14, as shown by complementation studies. The other two mutations—a -2 bp deletion at the same site, causing frameshift with a chain-terminating codon within a few triplets, and a base pair exchange at a nearby site-affect both the formation of 14 typical translation products and the trans-acting function. These results on box9 mutants combined with results on box7 mutants suggest that an 14-encoded “maturase” protein (apparent molecular weight, 27,000) is cleaved off a precursor protein (apparent molecular weight, 55,000) encoded by exon sequences B1 to B4 and the intron open reading frame. We further discuss the role of the box9 nucleotide sequence in the maturation of cob-specific RNA.     

Sequence analysis of mitochondrial DNA from Podospora anserina. Pervasiveness of a class I intron in three separate genes

A 48 kb region of the 95 kb mitochondrial genome of Podospora anserina has been mapped and sequenced (1 kb = 10(3) base-pairs). The DNA sequence of the genes for ND2, 3, 4, ATPase 6 and URFC are presented here. As in Neurospora crassa, the ND2 and 3 genes consist of a unit separated by one TAA stop codon. ND3, 4 and ATPase 6 are interrupted by class I introns. All three introns are remarkably similar in the C-domain of their secondary structure, sufficient enough to designate them as new subgroup, class IC introns. The open reading frames of the ND3 and 4 introns bear a high sequence similarity to the open reading frame of the class IB introns of ATPase 6 from N. crassa and ND1 from Neurospora intermedia Varkud. We also show that the tRNA Met-2 gene is duplicated and is involved in a recombinational event. The 5' region of URFC is also duplicated but no involvement of this gene with recombination or formation of plasmids is known. The evolutionary significance of the similarities of intron secondary structures and open reading frames of the ND3, 4 and ATPase 6 genes is discussed, including the possible separate evolution of structural and coding sequences.     

Molecular Characterization of the Mitochondrial DNA of a New Stopper Mutant Er-3 of Neurospora Crassa

An ethidium bromide-induced stopper mutant of Neurospora crassa is characterized at the molecular level. The mutant has two populations of mitochondrial DNA: a defective predominant mutant molecule and a basal level of the wild-type molecule. The aberrant DNA resulted after a 25-kbp deletion from the wild-type mitochondrial chromosome, which included major genes such as cytb, co1 and oli2. The deletion endpoints are located in the second intron of the ND5 gene, and in a sequence 250 nucleotides upstream of the co2 gene. The recombination has taken place between two nine nucleotide repeats CCCCGCCCC, one of which is close to a PstI palindrome at its 5' end. Thus the mutant ER-3 differs from all the other stopper mutants described previously in the extent and location of the deletions in the mtDNA.     

Cytochrome oxidase subunit II gene in mitochondria of Oenothera has no intron

The cytochrome oxidase subunit II gene has been localized in the mitochondrial genome of Oenothera berteriana and the nucleotide sequence has been determined. The coding sequence contains 777 bp and, unlike the corresponding gene in Zea mays, is not interrupted by an intron. No TGA codon is found within the open reading frame. The codon CGG, as in the maize gene, is used in place of tryptophan codons of corresponding genes in other organisms. At position 742 in the Oenothera sequence the TGG of maize is changed into a CGG codon, where Trp is conserved as the amino acid in other organisms. Homologous sequences occur more than once in the mitochondrial genome as several mitochondrial DNA species hybridize with DNA probes of the cytochrome oxidase subunit II gene.     

Universal code equivalent of a yeast mitochondrial intron reading frame is expressed into E. coli as a specific double strand endonuclease

The intron of the mitochondrial 21S rRNA gene of Saccharomyces cerevisiae (r1 intron) possesses a 235 codon long internal open reading frame (r1 ORF) whose translation product determines the duplicative transposition of that intron during crosses between intron-plus strains (omega+) and intron-minus ones (omega-). Using site-directed mutagenesis, we have constructed a universal code equivalent of the r1 ORF that, under appropriate promoter control, allows the overexpression in E. coli of a protein identical to the mitochondrial intron encoded "transposase". This protein exhibits a double strand endonuclease activity specific for the omega- site. This finding demonstrates, for the first time, the enzymatic activity of an intron encoded protein whose function is to promote the spreading of that intron by generating double strand breaks at a specific sequence within a gene.     

Interlocked RNA circle formation by a self-splicing yeast mitochondrial group I intron

RNA containing the aI3 group I intron of the yeast mitochondrial gene encoding cytochrome oxidase subunit I shows self-splicing in vitro. The excised intron, comprising 1514 nucleotides, is partially split into an upstream portion, containing the intronic reading frame, and a downstream portion, containing the typical group I conserved sequence elements. Full-length intron RNA and intron parts occur in linear and circular form. In the transesterification reactions leading to circle formation, only the guanosine nucleotide added during splicing is removed. Reincubation of isolated, complete circular intron RNA under self-splicing conditions leads to formation of free subintronic RNA circles. Under similar conditions, purified linear intron RNA gives rise to a number of circular and linear products, one of which consists of interlocked subintronic RNA circles. These observations suggest that the intron RNA possesses a dynamic structure in which subtle alterations in folding result in the formation of RNA products with different topology.