DNA sequence,structure, and phylogenetic relationship of the small subunit rRNA coding region of mitochondrial DNA fromPodospora anserina

Summary DNA sequence analysis and the localization of the 5 and 3 termini by S1 mapping have shown that the mitochondrial (mt) small subunit rRNA coding region fromPodospora anserina is 1980 bp in length. The analogous coding region for mt rRNA is 1962 bp in maize, 1686 bp inSaccharomyces cerevisiae, and 956 bp in mammals, whereas its counterpart inEscherichia coli is 1542 bp. TheP. anserina mt 16S-like rRNA is 400 bases longer than that fromE. coli, but can be folded into a similar secondary structure. The additional bases appear to be clustered at specific locations, including extensions at the 5 and 3 termini. Comparison with secondary structure diagrams of 16S-like RNAs from several organisms allowed us to specify highly conserved and variable regions of this gene. Phylogenetic tree construction indicated that this gene is grouped with other mitochondrial genes, but most closely, as expected, with the fungal mitochondrial genes.     

Characterization of a group I intron in the nuclera rDNA differentiatingPhialophora gregata f. sp.adzukicola fromP. gregata f. sp.sojae

An insertion sequence was detected near the 3′ end of the nuclear small subunit rDNA in isolates ofPhialophora gregata f. sp.adzukicola, the causal agent of the brown stem rot disease of adzuki bean. This insertion sequence was absent in isolates ofP. gregata, f. sp.sojae which causes brown stem rot of soybean. The insertion sequence is 304 bp long and contains all the characteristics of group I introns. These characteristics include, the four conserved sequence elements (P, Q, R, and S), a U at the 5′ splice site of the exon, a G at the 3′ splice site of the intron, a putative internal guiding sequences; the sequence also fits a secondary structure model for group I introns. Similar to most group I introns found in nuclear small subunit rDNA, the intron was located in a highly conserved region and is devoid of long open reading frames. This intron provides a convenient marker for use in conventional PCR to separateP. gregata f. sp.adzukicola fromP. gregata f. sp.sojae.     

Detection of a protein encoded by a class II mitochondrial intron of Podospora anserina

Summary In the filamentous fungus Podospora anserina, the amplification as circular DNA molecules of the first intron (intron ) of the CO1 mitochondrial gene, encoding the cytochrome oxidase subunit 1, is known to be strongly associated with aging of strains. In this study we have attempted to detect the protein potentially encoded by the open reading frame (ORF) contained in this intron. This was done by the Western blot technique using specific antisera raised against three polypeptides encoded by three non-overlapping fragments of this ORF adapted to the universal code and overexpressed in Escherichia coli. We examined about thirty independent subclones of Podospora derived from two different geographic races (A, s), using wild-type and mutant strains, young and senescent cultures. A 100 kDa polypeptide, encoded by the class II intron , was detected in five senescent subclones which all showed strong amplification of the intronic sequence (Sen DNA ).     

Evolutionary transfer of ORF-containing group I introns between different subcellular compartments (chloroplast and mitochondrion)

We describe here a case of homologous introns containing homologous open reading frames (ORFs) that are inserted at the same site in the large subunit (LSU) rRNA gene of different organelles in distantly related organisms. We show that the chloroplast LSU rRNA gene of the green alga Chlamydomonas pallidostigmatica contains a group I intron (CpLSU.2) encoding a site-specific endonuclease (I-CpaI). This intron is inserted at the identical site (corresponding to position 1931-1932 of the Escherichia coli 23S rRNA sequence) as a group I intron (AcLSU.m1) in the mitochondrial LSU rRNA gene of the amoeboid protozoon Acanthamoeba castellanii. The CpLSU.2 intron displays a remarkable degree of nucleotide similarity in both primary sequence and secondary structure to the AcLSU.m1 intron; moreover, the Acanthamoeba intron contains an ORF in the same location within its secondary structure as the CpLSU.2 ORF and shares with it a strikingly high level of amino acid similarity (65%; 42% identity). A comprehensive survey of intron distribution at site 1931 of the chloroplast LSU rRNA gene reveals a rather restricted occurrence within the polyphyletic genus Chlamydomonas, with no evidence of this intron among a number of non- Chlamydomonad green algae surveyed, nor in land plants. A parallel survey of homologues of a previously described and similar intron/ORF pair (C. reinhardtii chloroplast CrLSU/A. castellanii mitochondrial AcLSU.m3) also shows a restricted occurrence of this intron (site 2593) among chloroplasts, although the intron distribution is somewhat broader than that observed at site 1931, with site-2593 introns appearing in several green algal branches outside of the Chlamydomonas lineage. The available data, while not definitive, are most consistent with a relatively recent horizontal transfer of both site-1931 and site- 2593 introns (and their contained ORFs) between the chloroplast of a Chlamydomonas-type organism and the mitochondrion of an Acanthamoeba- like organism, probably in the direction chloroplast to mitochondrion. The data also suggest that both introns could have been acquired in a single event.      

Structure and expression of the overlapping ND4L and ND5 genes of Neurospora crassa mitochondria

Summary Genes homologous to the mammalian mitochondrial NADH dehydrogenase subunit genes ND4L and ND5 were identified in the mitochondrial genome of the filamentous fungus Neurospora crassa, and the structure and expression of these genes was examined. The ND4L gene (interrupted by one intervening sequence) potentially encodes an 89 residue long hydrophobic protein that shares about 26% homology (or 41% homology if conservative amino acid substitutions are allowed) with the analogous human mitochondrial protein. The ND5 gene (which contains two introns) encodes a 715 residue polypeptide that shares 23% homology with the human analogue; a 300 amino acid long region is highly conserved (50% homology) in the two ND5 proteins. The stop codon of the ND4L gene overlaps the initiation codon of the downstream ND5 gene, and the two genes are contranscribed and probably cotranslated. A presumed mature dicistronic (ND4L plus ND5) RNA was detected. The postulated mRNA (about 3.2 kb) contains 5 and 3 non-coding regions of about 86 and 730 nucleotides, respectively; this species is generated from very large precursor RNAs by a complex processing pathway. The ND4L and ND5 introns are all stable after their excision from the precursor species.Abbreviations bp base pairs - rRNA ribosomal RNA - ND NADH dehydrogenase - URF unidentified reading frame - kDal kilodaltons; a.a., amino acid     

Heterogeneous yet similar introns reside in identical positions of the rRNA genes in natural isolates of the archaeon Aeropyrum pernix

Some archaeal ribosomal DNA (rDNA) introns carry homing endonuclease-like genes and are therefore assumed to propagate by "intron homing". A previous study demonstrated that three introns are located within the rRNA operon (arnSL) of Aeropyrum pernix strain K1, two of which, Ialpha and Igamma, harbor open reading frames (ORFs) encoding putative LAGLIDADG-type endonucleases. In an effort to understand further the rDNA intron distribution in natural A. pernix populations, 11 A. pernix strains were isolated from marine hydrothermal biotopes, and comparative nucleotide sequence analysis of the arnSL alleles was performed. Of the 11 isolates, eight contained multiple introns, and three patterns of intron insertion were found. Three novel introns, Idelta (62 bp in length), Ivarepsilon (122 bp) and Izeta (57 bp) were identified. They were all ORF-less, but their predicted RNA secondary structure at the exon-intron junctions was consistent with the bulge-helix-bulge motif. The insertion positions and the terminal inverted repeat sequences of Idelta and Izeta were in agreement with those of Ialpha and Igamma, respectively. This suggests that these intron variants were generated by large indels (insertions/deletions) during their evolution.     

Eu-Chlorella large subunit rDNA sequences and group I introns in ribosomal DNA of the paramecian symbiotic alga NC64A

Although the examination of large subunit ribosomal RNA genes (LSU rDNA) is advanced in phylogenetic studies, no corresponding sequence data from trebouxiophytes have been published, with the exception of ‘Chlorella’ellipsoidea Gerneck. We determined the LSU rDNA sequence of Chlorella vulgaris Beijerinck and of the symbiotic alga of green paramecium, Chlorella sp. NC64A. A total of 59 nucleotide substitutions were found in the LSU rDNA of the two species, which are disproportionately distributed. Primarily, 65% of the substitutions were encountered in the first 800 bp of the alignment. This segment apparently has evolved eight times faster than the complete SSU rDNA sequence, making it a good candidate for a phylogenetic marker and giving a resolution level intermediate between small subunit (SSU) rDNA and internal transcribed spacers. Green algae are known as a group I intron‐rich group along with rhodophytes and fungi. NC64A is particularly rich in the introns; five introns were newly identified from the LSU rDNA sequence, which we named Cnc.L200, Cnc.L1688, Cnc.L1926, Cnc.L2184 and Cnc.L2437, following the insertion positions. In the present study we analyzed these introns with three others (Cnc.S943, Cnc.S1367 and Cnc.S1512) that had already been found in NC64A SSU rDNA. Secondary structure modeling placed these introns in the group I intron family, with four introns belonging to subgroup C1 and the other four introns belonging to subgroup E. Five of the intron insertion positions are unique to the paramecian symbiont, which may indicate relatively recent events of intron infections that includes transpositions. Intron phylogeny showed unprecedented relationships; four Cnc. IC1 introns made a clade with some green algal introns with insertions at nine different positions, whereas four Cnc. IE introns made a clade with the S651 intron (Chlorella sp. AN 1–3), which lay as a sister to the S516 insertion position subfamily.     

Occurrence of introns in the 16S rRNA genes of members of the genus Thermoproteus

Multiple introns were detected in the 16S rRNA gene of newly isolated Thermoproteus species strains IC-033 and IC-061 and Thermoproteus neutrophilus JCM 9278. In the 16S rRNA gene of strain IC-033, five introns of 627, 762, 636, 33, and 682 bp existed after positions 548, 781, 1092, 1205, and 1213 (according to the Escherichia coli numbering system), respectively. Likewise, strain IC-061 possessed 764-, 32-, and 688-bp introns after positions 781, 1205, and 1213, respectively; and T. neutrophilus JCM 9278 had 34- and 663-bp introns after positions 1205 and 1213, respectively. All the introns carried the putative intron core structures consisting of a bulge-helix-bulge motif and a long stable stem. The large introns carried open reading frames containing the LAGLI-DADG-like motifs in their terminal inserts; however, three out of four large introns of strain IC-033 seemed to incur frameshift mutations. Occurrence of introns at the same insertion sites in the three strains would allow tracing of the evolutionary movements of these introns. Received: 16 February 1998 / Accepted: 27 April 1998     

The mitochondrial genome of the fission yeast Schizosaccharomyces pombe. 2. Localization of genes by interspecific hybridization in strain ade7-50h- and cloning of the genome in small fragments

A series of 18 small overlapping restriction fragments has been cloned, covering the complete mitochondrial genome of Schizosaccharomyces pombe. By hybridizing mitochondrial gene probes from Saccharomyces cerevisiae and Neurospora crassa with restriction fragments of Schizosaccharomyces pombe mitochondrial DNA, the following homologous genes were localized on the mitochondrial genome of S. pombe: cob, cox1, cox2 and cox3, ATPase subunit 6 and 9 genes, the large rRNA gene and both types of open reading frames occurring in mitochondrial introns of various ascomycetes. The region of the genome, hybridizing with cob exon probes is separated by an intervening sequence of about 2500 bp, which is homologous with the first two introns of the cox1 gene in Saccharomyces cerevisiae (class II introns according to Michel et al. 1982). Similarly, in the cox1 homologous region, which covers about 4000 bp, two regions were detected hybridizing with class I intron probes, suggesting the existence of two cox1 introns in Schizosaccharomyces pombe. Hybridization with several specific exon probes with a determined order has revealed that cob, cox1, cox3 and the large rRNA gene are all transcribed from the same DNA strand. The low intensities of hybridization signals suggest a large evolutionary distance between Schizosaccharomyces pombe and Saccharomyces cerevisiae or Neurospora crassa mitochondrial genes. Considering the length of the mitochondrial DNA of Schizosaccharomyces pombe (about 19.4 kbp) and the expected length of the localized genes and intron sequences there is enough space left for encoding the expected set of tRNAs and the small rRNA gene. The existence of leader-, trailer-, ori- and spacer sequences or further unassigned reading frames is then restricted to a total length of about 3000 bp only.     

The mosaic cox1 gene in the mitochondrial genome of Schizosaccharomyces pombe: minimal structural requirements and evolution of group I introns

The gene encoding subunit 1 of cytochrome oxidase (cox1) in the fission yeast Schizosaccharomyces pombe is polymorphic. In strain 50 it contains two group I introns with open reading frames (ORFs) in phase with the upstream exons (Lang, 1984). In strain EF1 two additional very short group I introns which do not possess ORFs were detected by DNA sequencing. These two introns (AI2a and AI3) share distinct characteristics concerning their nucleotide sequence and secondary structure and are located at identical positions as the introns AI4 and AI5 beta, respectively, in the cox1 gene of Saccharomyces cerevisiae. The sequence homology of the cob and cox1 genes around the splice points of introns AI2a, AI4, and BI4 (cob intron 4) might reflect horizontal gene transfer between the distantly related species S. pombe and S. cerevisiae.     

The intron of the mitochondrial 21S rRNA gene: distribution in different yeast species and sequence comparison between Kluyveromyces thermotolerans and Saccharomyces cerevisiae

We have screened numerous different yeast species for the presence of sequences homologous to the intron of the mitochondrial 21S rRNA gene of Saccharomyces cerevisiae (intron r1) and found them in all Kluyveromyces species, some of the Saccharomyces species and none of the other yeasts tested. We have determined the nucleotide sequence of the r1-intron in K. thermotolerans and compared it with that of S. cerevisiae. The two introns are inserted at the same position within the 21S rRNA gene. They contain homologous internal open reading frames (ORFs) initiated at the same AUG codon which can be aligned over their entire length. Several silent multi-substitutions indicate that these intronic ORFs represent selectively conserved functional genes. Other intron segments, on the contrary, reveal short blocks of extensive homology separated by non-homologous stretches and/or additions-deletions. Comparison of our two yeast r1-introns with equivalent introns of N. crassa and A. nidulans mitochondria reveals that introns with very similar RNA secondary structures can accommodate different types of ORFs.     

Cloning and characterization of the rDNA repeat unit of Podospora anserina

Summary DNA coding for ribosomal RNA in Podospora anserina has been cloned and was found as a tandemly repeated 8.3 kb sequence. The cloned rDNA was characterized by restriction endonuclease mapping. The location of 5.8S, 18S and 28S rRNA coding regions was established by DNA-RNA hybridization and S1 nuclease mapping. The organization of P. anserina rRNA genes is similar to that of Neurospora crassa and Aspergillus nidulans. The rDNA unit does not contain the sequence coding for 5S RNA.     

Using bacteria to analyze sequences involved in chloroplast gene expression

The complete nucleotide sequence of chloroplast DNA from a liverwort, Marchantia polymorpha has made clear the entire gene organization of the chloroplast genome. Quite a few genes encoding components of photosynthesis and protein synthesis machinery have been identified by comparative computer analysis. Other genes involved in photosynthesis, respiratory electron transport, and membrane-associated transport in chloroplasts were predicted by the amino acid sequence homology and secondary structure of gene products. Thirty-three open reading frames in the liverwort chloroplast genome remain unidentified. However, most of these open reading frames are also conserved in the chloroplast genomes of two species, a liverwort, Marchantia polymorpha, and tobacco, Nicotiana tabacum, indicating their active functions in chloroplasts.Abbreviations bp base pair - kDa kilodalton - IR inverted repeat - ORF open reading frame - DALA -aminolevulinate     

The untranslated leader of nuclear COX4 gene of Saccharomyces cerevisiae contains an intron.

The nuclear gene for subunit IV of cytochrome oxidase (COX4) in Saccharomyces cerevisiae contains a 342 bp intron which is contained entirely within the 5' leader of the message. Splicing of the intron results in removal of several small open reading frames; subsequently, the COX4 AUG becomes the 5' proximal initiation codon. A strain with an rna2- mutation fails to splice mRNA efficiently at restrictive temperature and was used to map the intron splice junctions by RNase protection. Two major mRNA initiation sites were mapped by primer extension of synthetic oligodeoxynucleotides. The splice junctions and internal TACTAAC box conform to consensus sequences previously determined from other yeast introns. One gene for subunit V of cytochrome oxidase (COX5b) has also been shown to contain an intron. The significance of introns in two nuclear genes encoding subunits of cytochrome oxidase is discussed.     

Three variant introns of the same general class in the mitochondrial gene for cytochrome oxidase subunit 1 in Aspergillus nidulans

The oxiA gene of Aspergillus nidulans, coding for cytochrome oxidase subunit 1, is shown by DNA sequencing to contain three introns. An AUG start codon is not present at the beginning of the sequence, suggesting that either another codon, possibly the four base codon AUGA, is used for initiation or there is a further short intron between the true start codon and the beginning of the recognisable coding region. The second and third introns have long open reading frames, which could code for maturase proteins. The lack of conservation of amino acid sequence in the putative region of proteolytic cleavage for maturase formation suggests that the first conserved decapeptide may act as the recognition signal for protein processing. The third intron is remarkably (70%) homologous to the second intron of the cytochrome oxidase subunit 1 gene of Schizosaccharomyces pombe and both are located in exactly the same position. The third Aspergillus intron has an in-frame insertion of a 37-bp GC-rich DNA sequence which is now flanked by a 5-bp repeat, a well-known feature of transposable elements. All three introns in the oxiA gene have a 'core' RNA secondary structure found in a class of introns fitting the RNA splicing model of Davies et al. (1982). This core RNA structure may play a catalytic as well as a structural role in intron splicing. A sequence within the intron could act as a guide to align the splice sites of two of the introns in accordance with the model of Davies et al.