DNA sequence analysis of the apocytochrome b gene of Podospora anserina: a new family of intronic open reading frame

Summary The 5,969 by (base pair) DNA sequence of the apocytochrome b mitochondrial (mt) gene of race A Podospora anserina was located in a 8.5 Kbp region. This gene contained a 2,499 by subgroup IB and a 1,306 by subgroup ID intron as well as a 990 bp subgroup IB intron which is present in race A but not race s. The large subgroup IB intron and the race A specific IB intron both contained potential alternate splice sites which brought their open reading frames into phase with their upstream exon sequences. All three introns were compared with regard to their secondary structures and open reading frames to the other 30 group I introns in Podospora anserina, as well as to other fungal introns. We detected a new family of intronic ORFs comprising seven P. anserina introns, several N. crassa introns, as well as the T4td bacteriophage intron. Sequence similarities to intron-encoded endonucleases were noteworthy. The DNA sequences reported here and in the accompanying paper complete the analysis of race s and race A mitochondrial DNA.     

DNA sequence analysis of the 24.5 Kilobase pair cytochrome oxidase subunit I mitochondrial gene from Podospora anserina: a gene with sixteen introns

Summary The DNA sequence of a 26.7 Kilobase pair (10³ base pairs = 1 Kb) region of the mitochondrial genomes of races s and A from Podospora anserina was determined. Within this region, the 24.5 Kb cytochrome oxidase subunit I gene was located and its exon sequences determined by computer analysis comparisons with other fungal genes. The Podospora COI gene was interrupted by two group II introns (one in race s) and fourteen group I introns ranging in size from about 2.2 Kb to 404 bp. Earlier studies on secondary structure analysis, as well as comparison of their open reading frames (ORFs), showed that the two group II introns were closely related. The fourteen group I introns were representatives of three subgroupings (IB, C and a new category, subgroup ID). Two of these group I introns were separated by just a single exon codon. The analysis of all these introns is discussed in comparison with other fungal introns as well as with the known Podospora anserina introns.     

Distribution of mitochondrial introns in the species Schizosaccharomyces pombe and the origin of the group II intron in the gene encoding apocytochrome b

Summary The mitochondrial genome size of 26 different Schizosaccharomyces pombe strains varies between 17.6 and 24.6 kilobase pairs due to the presence or absence of introns. One of these is the group II intron in the gene encoding apocytochrome b (cob: intron cobI1 ). Partial DNA sequences of continuous cob genes from six strains (including strain EF1: Trinkl et al. 1985) revealed identical nucleotide sequence in the region where the group II intron is inserted in the mosaic form of the gene. In contrast, analysis of the mosaic cob, gene in strain UCD-Fstl revealed several base pair changes in the exon regions flanking the splice point, compared with the continuous genes and with the mosaic cob gene in strain 50 (Lang et al. 1985). The base pair differences between the exons of the two mosaic cob genes and the identity of exons in all continuous cob genes argue in favour of the two cob introns in strains 50 and UCD-FstI as independent later acquisitions of the genes, rather than loss of the intron from a common mosaic ancestor of all strains.Other introns present in some but not all strain include two group I introns without open reading frame in the gene encoding subunit 1 of cytochrome c oxidase (cox1: introns cox1I2a and cox1I3), and two group I introns with open reading frames in the same gene (introns cox1I1 and cox1I2b).     

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.     

The Chlorella H+/hexose cotransporter gene

Summary The complete genomic sequence of the inducible Chlorella kessleri H⁺/hexose cotransporter (HUP1) has been obtained from two overlapping clones isolated from a gt10 library. The HUP1 gene is interrupted by 14 introns with the first intron being located in the 5-untranslated part of the gene. The average intron length is 220 bp, yielding a very regular intron/exon pattern in the gene. The codon usage in this gene is strongly biased with a clear preference for C and a strong suppression of A. A consensus sequence for a putative algal polyadenylation sequence is shown and compared with other algal cDNA sequences.     

The complete DNA sequence of the mitochondrial genome of Podospora anserina

Summary The complete 94,192 bp sequence of the mitochondrial genome from race s of Podospora anserina is presented (1 kb=10³ base pairs). Three regions unique to race A are also presented bringing the size of this genome to 100,314 bp. Race s contains 31 group I introns (33 in race A) and 2 group II introns (3 in race A). Analysis shows that the group I introns can be categorized according to families both with regard to secondary structure and their open reading frames. All identified genes are transcribed from the same strand. Except for the lack of ATPase 9, the Podospora genome contains the same genes as its fungal counterprts, N. crassa and A. nidulans. About 20% of the genome has not yet been identified. DNA sequence studies of several excision-amplification plasmids demonstrate a common feature to be the presence of short repeated sequences at both termini with a prevalence of GGCGCAAGCTC.     

Analysis of class I introns in a mitochondrial plasmid associated with senescence of Podospora anserina reveals extraordinary resemblance to the Tetrahymena ribosomal intron

Summary Recently, the nucleotide sequences for three mitochondrial plasmids associated with senescence of Podospora anserina were determined (Cummings et al. 1985). One of these sequences, corresponding to the plasmid termed senDNA, contains three class I introns, all within a protein coding sequence equivalent to the mammalian URF1 gene. Here, we present primary and secondary structure analyses for two of these introns as well as a partial analysis for the third, which extends beyond the DNA sequence determined. With regard to both primary and secondary structure, the closest known relative of intron 1 is the self-splicing intron in the large ribosomal RNA gene of Tetrahymena. One secondary structure domain at the periphery of intron 1 and Tetrahymena models is also present in intron 2. The latter intron is the longest known class I member and contains remnants of two protein-coding sequences, one of which is split by the other. Evolutionary processes that might be responsible for the unusual structure of introns 1 and 2 are discussed.     

Two self-splicing group I introns interrupt two late transcribed genes of prolate-headed <Emphasis Type="Italic">Lactobacillus delbrueckii</Emphasis> phage JCL1032

Summary. Two group I introns were detected from the late gene region of the prolate-headed phage JCL1032 of Lactobacillus delbrueckii. Introns JCL-I1 and JCL-I2 interrupt orf602 and orf1868 encoding a phage terminase large subunit (TerL, 69.7kDa) and a putative tape measure protein (TMP, 202kDa), respectively. The introns JCL-I1 (226bp) and JCL-I2 (322bp) were efficiently self-spliced in vivo. Both introns were classified to the subgroup IA1 having all the conserved structures necessary for splicing, but lacking the ability to encode endonucleases or other gene products. The introns JCL-I1 and JCL-I2 shared restricted nucleotide sequence similarity with each other and with the group I terL intron of Lb. delbrueckii phage LL-H. No match was found for JCL-I1 in the homology searches. Instead, the primary sequence from the joining region of P8 and P7 to P9 of the intron JCL-I2 was homologous to the group I intron of Bacillus mojavensis; the orf142 introns I1, I2 and I3 of Staphylococcus aureus phage Twort; the group I intron of phage Bastille (Bacillus thuringiensis); and to the group IA3 intron of Monomastix species.     

The catalytic group-I introns of the psbA gene of Chlamydomonas reinhardtii : core structures, ORFs and evolutionary implications

The sequences and predicted secondary structures of the four catalytic group-I introns in the psbA gene of Chlamydomonas reinhardtii, Cr.psbA-1–Cr.psbA-4, have been determined. Cr.psbA-1 and Cr.psbA-4 are subgroup-IA1 introns and have similar secondary structures, except at the 3′ end where Cr.psbA-1 contains a large inverted-repeat domain. Cr.psbA-4 is closely related to intron 1 of the Chlamydomonas moewusii psbA gene, with which it shares the same location, high nucleotide identity in the core, and an identically placed ORF that shows 58% amino-acid identity. Cr.psbA-2 is a subgroup-IA3 intron, and shows similarities to the Chlamydomonas eugametos rRNA intron, Ce.LSU-1. Cr.psbA-3 is a subgroup-IA2 intron, and is remarkably similar to the T4 phage intron, sunY. Interestingly, a degenerate version of Cr.psbA-3 is located in the intergenic region between the chloroplast petA and petD genes. All four introns contain ORFs, which potentially code for basic proteins of 11–38 kDa. The ORFs in introns 2 and 3 contain variants of the GIY-YIG motif; however, the Cr.psbA-2 ORF is free-standing, whereas the Cr.psbA-3 ORF is contiguous and in-frame with the upstream exon. The Cr.psbA-4 ORF contains an H-N-H motif, and possibly a GIY-YIG motif. These data indicate that the C. reinhardtiipsbA introns have multiple origins, and illustrate some of the evolutionary DNA dynamics associated with group-I introns in Chlamydomonas. Received: 24 November 1998 / 23 March 1999     

Nucleotide-sequence analysis indicates that a DNA plasmid in a diseased isolate of Ophiostoma novo-ulmi is derived by recombination between two long repeat sequences in the mitochondrial large subunit ribosomal RNA gene

The nucleotide sequence of a mitochondrial plasmid (2234 bp) in a diseased isolate of Ophiostoma novo-ulmi, and sequences of the mitochondrial DNA that overlap and flank the plasmid end-points, have been determined. The plasmid was shown to be derived from the O. novo-ulmi mitochondrial large subunit ribosomal RNA gene and contained most of intron 1, the whole of exon 2, and probably the first part of intron 2. Within intron 1 there is an open reading frame with the potential to encode a 323 amino-acid polypeptide which contained dodecapeptide sequences typical of RNA maturases and DNA endonucleases. The endpoints of the plasmid in the mtDNA were located within two 90-bp direct imperfect repeat sequences, one of which comprised the last 7 bp of exon 1 and the first 83 bp of intron 1 whilst the other comprised the last 7 bp of exon 2 and the first 83 bp of intron 2. It is proposed that the Ld plasmid was generated by intramolecular recombination between these two repeats with the crossover point probably within the last 15 bp.     

Three mitochondrial unassigned open reading frames of Podospora anserina represent remnants of a viral-type RNA polymerase gene

The mitochondrial DNA of Podospora anserina is complex, consisting of a characteristic set of genes with a large number of introns and a substantial amount of sequence of unknown function and origin. In addition, as indicated by various types of reorganization, this genome is highly flexible. Here we report the identification of three unassigned mitochondrial open reading frames (ORF P', ORF Q', ORF 11) as remnants of a rearranged viral-type RNA polymerase gene. These ORFs are not transcribed and may be derived from the integration of a linear plasmid of the type recently identified in a mutant of P. anserina.     

Characterization of a genomic region encoding the 32-kDa dense granule antigen of Sarcocystis muris (Apicomplexa)

Two subgenomic libraries constructed from Sarcocystis muris total DNA were screened with a cDNA probe, specific for a 32-kDa protein associated with the dense granules. Two clones reacted positively and were isolated, gDG 32/1 and gDG 32/2. Genomic clone gDG32/1 and part of clone gDG 32/2 have been sequenced. The composite nucleotide sequence of these genomic clones comprises 4.34 kb. It contains a 5′ region of 2.14 kb, a first coding region (222 bp, exon I), a noncoding region (608 bp, intron), a second coding region (660 bp, exon II), and a 3′ region of 693 bp. The upstream region shows a eukaryotic promoter structure and a consensus sequence for the 5′ and 3′ splicing sites. Thus the open reading frame (ORF DG32) coding for the 32-kDa protein of the dense granules of S. muris cyst merozoites is interrupted by an intron. To our knowledge, dg32 is the first sarcosporidian mosaic gene to be characterized. Received: 27 April 1999 / Accepted: 11 May 1999     

Molecular cloning of thi-4, a gene necessary for the biosynthesis of thiamine in Neurospora crassa

The thiamine-4 (thi-4) gene was cloned by functional complementation of a thi-4 mutant of Neurospora crassa. The product of this gene is believed to be involved in the condensation of pyrimidine and thiazole precursors, which is necessary for the synthesis of thiamine. The thi-4 gene has ten introns which vary in length from 57 to 200 bp and the junction and internal (lariat) sequences are in good agreement with the consensus sequences for the splicing of introns in N. crassa. The thi-4 gene encodes a protein of 538 amino acids which is similar in terms of amino-acid sequence to proteins encoded by Saccharomyces cerevisiae whose function is unknown. The expression of the thi-4 gene in N. crassa was not repressed by thiamine. Received: 19 December 1995 / Accepted: 16 January 1996     

Self-splicing activity of the mitochondrial group-I introns from Aspergillus nidulans and related introns from other species

The mitochondrial genome of Aspergillus nidulans contains several group-I introns. Each one has been assayed for its ability to self-splice in vitro in the absence of proteins. The intron from the apocytochrome b gene is unusual among subgroup IB4 introns in being able to self-splice, unlike a similar intron from Saccharomyces cerevisiae. The first intron in the cytochrome oxidase subunit-1 gene self-splices but only correctly completes the first step of splicing; cryptic 3′ splice-sites are recognized instead and these are also used at a low frequency in vivo. The highly homologous intron from Podospora anserina completes both steps in vitro. The remaining introns do not self-splice. The correlation between subgroup category, the likely presence of specific tertiary interactions, and self-splicing activity is discussed. Received: 16 May / 25 August 1997     

The linear mitochondrial plasmid pAL2-1 of a long-lived Podospora anserina mutant is an invertron encoding a DNA and RNA polymerase

The molecular characterization of an additional DNA species (pAL2-1) which was identified previously in a long-lived extrachromosomal mutant (AL2) of Podospora anserina revealed that this element is a mitochondrial linear plasmid. pAL2-1 is absent from the corresponding wild-type strain, has a size of 8395 bp and contains perfect long terminal inverted repeats (TIRs) of 975 bp. Exonuclease digestion experiments indicated that proteins are covalently bound at the 5 termini of the plasmid. Two long, non-overlapping open reading frames, ORF1 (3,594 bp) and ORF2 (2847 bp), have been identified, which are located on opposite strands and potentially encode a DNA and an RNA polymerase, respectively. The ORF1-encoded polypeptide contains three conserved regions which may be responsible for a 3–5 exonuclease activity and the typical consensus sequences for DNA polymerases of the D type. In addition, an amino-acid sequence motif (YSRLRT), recently shown to be conserved in terminal proteins from various bacteriophages, has been identified in the amino-terminal part of the putative protein. According to these properties, this first linear plasmid identified in P. anserina shares all characteristics with invertrons, a group of linear mobile genetic elements.     

Nucleotide sequence of the COX1 gene in Kluyveromyces lactis mitochondrial DNA: evidence for recent horizontal transfer of a group II intron

Summary The cytochrome oxidase subunit 1 gene (COX1) in K. lactis K8 mtDNA spans 8 826 bp and contains five exons (termed E1–E5) totalling 1 602 bp that show 88% nucleotide base matching and 91% amino acid homology to the equivalent gene in S. cerevisiae. The four introns (termed K1 cox1.1–1.4) contain open reading frames encoding proteins of 786, 333, 319 and 395 amino acids respectively that potentially encode maturase enzymes. The first intron belongs to group II whereas the remaining three are group I type B. Introns K1 cox1.1, 1.3, and 1.4 are found at identical locations to introns Sc cox1.2, 1.5a, and 1.5b respectively from S. cerevisiae. Horizontal transfer of an intron between recent progenitors of K. lactis and S. cerevisiae is suggested by the observation that K1 cox1.1 and Sc cox1.2 show 96% base matching. Sequence comparisons between K1 cox1.3/Sc cox1.5a and K1 cox1.4/Sc cox1.5b suggest that these introns are likely to have been present in the ancestral COX1 gene of these yeasts. Intron K1 cox1.2 is not found in S. cerevisiae and appears at an unique location in K. lactis. A feature of the DNA sequences of the group I introns K1 cox1.2, 1.3, and 1.4 is the presence of 11 GC-rich clusters inserted into both coding and noncoding regions. Immediately downstream of the COX1 gene is the ATPase subunit 8 gene (A8) that shows 82.6% base matching to its counterpart in S. cerevisiae mtDNA.EM BL Accession Number X57546     

Messenger RNA intron in the nuclear 18s ribosomal RNA gene of deuteromycetes

Introns within messenger RNA genes have characteristic border sequences and a conserved region near the 3 end of the intron. All are involved in splicing to produce the mature mRNA. Introns in ribosomal RNA genes have less well-defined borders and contain no internal conservation. We report here mRNA-type introns located near the 3 end of the 18s rRNA genes of the deuteromycetes Phialophora americana and Cenococcum geophilum. Inserted sequences of various sizes have also been located at the same point in several other deuteromycete species.     

Sequence of the nuclear ATP synthase subunit 9 gene of Podospora anserina: lack of similarity to the mitochondrial genome

Summary The nuclear gene coding for the mitochondrial subunit 9 of the F₀F₁-ATP synthase complex was isolated from a genomic library of Podospora anserina. Nucleotide sequencing revealed an open reading frame capable to code for 144 amino acids including an amino-terminal pre-sequence of 63 amino acid residues for mitochondrial import of the pre-proteolipid. The P. anserina proteolipid shows extensive sequence identity with the corresponding gene products of the related filamentous fungi Neurospora crassa, Aspergillus nidulans and Aspergillus niger. In contrast to the situation in Saccharomyces cerevisiae, N. crassa and A. nidulans, no sequence similarity of the ATP synthase subunit 9 gene to the mitochondrial genome of P. anserina could be detected. Thus, in P. anserina this gene appears to be exclusively encoded by the nuclear genome.     

Polymorphism of the mitochondrial L-RNA gene of the basidiomycete Coprinus cinereus

Summary Two unexpectedly small mitochondrial (mt) genomes of Coprinus cinereus, P and S, were compared with the H and J genomes we have described previously. H and J are 42 kb in size and differ in having alternative 1.23 kb insertions in or adjacent to the co-1 gene. P and S DNAs lacked both insertions and had an identical 4.4 kb deletion between the co-1 and L-RNA gene. P DNA contained a 700 by insertion and S DNA a 300 by deletion within a sequence coding the L-RNA gene. This was shown by Southern blot analysis using probes containing the 5 or the 3 exon sequences of the L-RNA gene of Neurospora crassa. These hybridisations showed also that the L-RNA gene and co-1 gene in the C. cinereus mt genome are oppositely orientated and must be transcribed from different DNA strands. No DNA homology was detected using probes containing intron sequences from the L-RNA genes of Saccharomyces cerevisiae or N. crassa. There was no evidence of respiratory deficiency in P and S strains and transfer of nuclei by dikaryon formation made it possible to recombine H nuclei with P and S mitochondria, S nuclei with H and P mitochondria and P nuclei with H mitochondria with no apparent detrimental effect on growth. We conclude that P and S mtDNAs represent naturally occurring variants of the C. cinereus mt genome.