The mitochondrial genome of fission yeast: inability of all introns to splice autocatalytically, and construction and characterization of an intronless genome

Summary In this paper we report the inability of four group I introns in the gene encoding subunit I of cytochrome c oxidase (cox1) and the group II intron in the apocytochrome b gene (cob) to splice autocatalytically. Furthermore we present the characterization of the first cox1 intron in the mutator strain ana ^r -14 and the construction and characterization of strains with intronless mitochondrial genomes. We provide evidence that removal of introns at the DNA level (termed DNA splicing) is dependent on an active RNA maturase. Finally we demonstrate that the absence of introns does not abolish homologous mitochondrial recombination.Abbreviations cox1, cox2, cox3 genes encoding subunits 1, 2 and 3 of cytochrome - c oxidase - cob gene encoding apocytochrome b - cox1I1, cox1I2a, cox1I2b, cox1I3 introns in cox1 - cox1Ix ^+/– indicates the presence or absence of the intron either in the native gene or after intron DNA excision - cox1Ix is a deletion in the intron leading to respiratory deficiency     

The apocytochrome b gene of Chlamydomonas smithii contains a mobile intron related to both Saccharomyces and Neurospora introns.

Summary The mitochondrial DNA of the two interfertile algal species Chlamydomonas smithii and Chlamydomonas reinhardtii are co-linear with the exception of ca. 1 kb insertion (the a insert) present in C. smithii DNA only. In vegetative diploids resulting from interspecific crosses, mitochondrial genomes are transmitted biparentally except for the a insert which is transmitted to all C. reinhardtii molecules in a manner reminiscent of the intron-mediated conversion event that occurs at the omega locus in yeast mitochondria, under the action of the I-SceI endonuclease. Here we report that the insert corresponds to a typical group I intron of 1075 bp, inserted within the gene for apocytochrome b and containing a 237 codon open reading frame (ORF). We also report the complete sequence of the apocytochrome b gene of C. smithii. Comparison with the sequence of the same gene in C. reinhardtii reveals the precise intron insertion site. These data, together with the previous genetic data provide the first example of intron mobility in mitochondria of the plant kingdom. The product of the intronic ORF shows 36% amino acid identity with the I-SceI endonuclease whereas the intron ribozyme shows a 60% identity at the nucleotide level with the Neurospora crassa cob · 1 intron. The possibility of a recent horizontal transfer of introns between fungi and algae is discussed.     

Isolation of a gene (pbsC) required for siderophore biosynthesis in fluorescent Pseudomonas sp. strain M114

An iron-regulated gene, pbsC, required for siderophore production in fluorescent Pseudomonas sp. strain M114 has been identified. A kanamycin-resistance cassette was inserted at specific restriction sites within a 7 kb genomic fragment of M114 DNA and by marker exchange two siderophore-negative mutants, designated M1 and M2, were isolated. The nucleotide sequence of approximately 4 kb of the region flanking the insertion sites was determined and a large open reading frame (ORF) extending for 2409 by was identified. This gene was designated pbsC (pseudobactin synthesis C) and its putative protein product termed PbsC. PbsC was found to be homologous to a family of enzymes involved in the biosynthesis of secondary metabolites, including EntF of Escherichia coli. These enzymes are believed to act via ATP-dependent binding of AMP to their substrate. Several areas of high sequence homology between these proteins and PbsC were observed, including a conserved AMP-binding domain. The expression of pbsC is iron-regulated as revealed when a DNA fragment containing the upstream region was cloned in a promoter probe vector and conjugated into the wild-type strain, M114. The nucleotide sequence upstream of the putative translational start site contains a region homologous to previously defined –16 to –25 sequences of iron-regulated genes but did not contain an iron-box consensus sequence. It was noted that inactivation of the pbsC gene also affected other iron-regulated phenotypes of Pseudomonas M114.     

DNA splicing of mitochondrial group I and II introns in Schizosaccharomyces pombe

Summary In this paper we report the precise excision of the group I intron aI2b from the cox1 gene and of the group II intron bI from the cob gene fo the Schizosaccharomyces pombe strain 50. We present evidence that DNA excision of both intron DNA sequences is under nuclear control. Attempts to remove the first cox1 intron (aI1) have failed so far, but a deletion of approximately 200 bp in the open intronic reading frame demonstrates that it is not essential for normal cellular functions.Abbreviations cox1, cox2, cox3 genes encoding subunits 1, 2 and 3 of cytochrome c oxidase - cob gene encoding apocytochrome b - rns and rnl genes encoding the small and large ribosomal RNA - atp6, atp8 and atp9 genes encoding subunits 6, 8, and 9 of the ATP synthase complex - urfa unassigned reading frame a - aI1, aI2a, aI2b, aI3 introns in the cox 1 gene of S. pombe - bI intron in the cob gene - del-aI2b and del-bI respiratory competent strains in which the respective introns have been deleted by DNA splicing     

Cytochrome b of cob revertants in yeast. 1. Isolation and characterization of revertants derived from cob exon mutants of Saccharomyces cerevisiae

Summary About 300 revertants were derived from 44 cob ^- mutants, mapping in the structure coding regions (exon 1, 3, 4, 5, or 6) of the mitochondrial apocytochrome b gene in Saccharomyces cerevisiae, strain 777-3A. Most of the revertants could not be distinguished from the wild-type by means of physiological properties. Twenty-two revertants different in phenotype are described here in more detail.The suppressor mutations (sup ^a) that compensate the primary cob ^- mutations (i.e., restore growth on glycerol) are mitochondrially inherited. They were localized in the same cob exon regions as the respective primary mutations, except for one revertant with a primary mutation in exon 6 and a suppressor, 4.2 map units distant, which may be located either in intron 5 or downstream in exon 6.Of 21 suppressors 17 are closely coupled to the primary mutation with recombination frequencies of 0.1%–0.3%. An estimate predicts that in more than 80% of these revertants only one amino acid is altered at that point of the polypeptide corresponding to the cob ^- site in the gene.The most interesting revertant phenotypes are: (1) reduced growth rate on glycerol. The respective cob ^-/sup^a mutations are scattered over the whole cob region and cannot be correlated exclusively with special gene regions. (2) decreased cytochrome b content. The most extreme reductions (28% and 30% of wild-type level) were observed to be due to mutations located in the 5 proximal part of exon 1. The highest percentage of revertants with decreased cytochrome b content was predominantly found mapping in exon 3. Complications in protoporphyrin attachment or the chelatase reaction were assumed to be the basic lesion causing reduced cytochrome b content, since in 10 out of 11 revertants examined the polypeptide is produced at wild-type level. (3) shifted maximum absorption wavelength of cytochrome b. The double mutations of the respective revertants map in the middle part of exon 1, in exon 4 and exom 5. The corresponding regions in the polypeptide presumably surround the heme group.     

A deletion in the sapA homologue cluster is responsible for the loss of the S-layer in Campylobacter fetus strain TK

The surface array protein (SAP) of Campylobacter fetus strain TK is encoded by seven homologous sapA genes clustered on the chromosomal DNA. The spontaneously arising variant strain TK(SAP^–) produces no SAP and carries an approximately 10-kb chromosomal deletion. To elucidate the mechanism underlying the loss of SAP synthesis, we analyzed the region containing the sapA homologues and the deletion. We constructed a physical map of the sapA cluster region by aligning the clones that contain sapA homologues. These analyses demonstrated that all sapA homologues were located within a limited region of about 50 kb of chromosomal DNA of strain TK. The TK(SAP^–) deletion was located within this cluster and was 13.3 kb in size. The deletion occurred between two sapA homologues and resulted in the formation of a chimeric sapA homologue in the variant strain. Sequence analysis of the upstream regions and the conserved regions of all sapA homologues revealed a high degree of similarity. However, only one sapA homologue contained a putative promoter sequence. This promoter sequence was located in the deleted region. Thus, the deletion of the promoter appears to be responsible for the loss of SAP expression in TK(SAP^–). Received: 17 May 1996 / Accepted: 6 December 1996     

A new repetitive sequence uniquely present in the decay-accelerating factor genes

 The decay-accelerating factor (DAF, CD55) protects cells from autologous complement attack on self cell membranes. We have previously reported that the seventh exon encoding the serine/threonine-rich(S/T)-abc region of the guinea pig DAF gene is composed of five homologous repeats of about 51 base pairs, and that differential usage of these repeats produces the various lengths observed in the S/T region of guinea pig DAF. In this study, we found that the seventh intron of the guinea pig DAF gene was wholly composed of 18 tandem repeats homologous to the repeating unit of the S/T-abc exon. This type of repetitive structure, although the number of repeats was variable, was also found in the corresponding exons and introns of all DAF genes of other species so far tested including human and seven other primates and mouse, in which alternative splicing in this region has not been found. This suggested that generation of the repetitive sequences spanning the exon and intron regions had occurred before the diversification of these species. In addition, all the intron sequences of the tested DAF genes had no stop codon when they were presumably translated in the same reading frame as the seventh and eighth exons, except for that of one of two duplicated mouse DAF genes. These findings and significant interspecies identities of the intron sequence suggest that the intron sequence conceivably could be translated in some tissues and/or in some stages of development although to date we have not yet succeeded in detecting mRNA for this region. Received: 7 July 1997 / Revised: 11 August 1997     

Isolation of a gene (pbsC) required for siderophore biosynthesis in fluorescent Pseudomonas sp. strain M114

An iron-regulated gene, pbsC, required for siderophore production in fluorescent Pseudomonas sp. strain M114 has been identified. A kanamycin-resistance cassette was inserted at specific restriction sites within a 7 kb genomic fragment of M114 DNA and by marker exchange two siderophore-negative mutants, designated M1 and M2, were isolated. The nucleotide sequence of approximately 4 kb of the region flanking the insertion sites was determined and a large open reading frame (ORF) extending for 2409 by was identified. This gene was designated pbsC (pseudobactin synthesis C) and its putative protein product termed PbsC. PbsC was found to be homologous to a family of enzymes involved in the biosynthesis of secondary metabolites, including EntF of Escherichia coli. These enzymes are believed to act via ATP-dependent binding of AMP to their substrate. Several areas of high sequence homology between these proteins and PbsC were observed, including a conserved AMP-binding domain. The expression of pbsC is iron-regulated as revealed when a DNA fragment containing the upstream region was cloned in a promoter probe vector and conjugated into the wild-type strain, M114. The nucleotide sequence upstream of the putative translational start site contains a region homologous to previously defined ?16 to ?25 sequences of iron-regulated genes but did not contain an iron-box consensus sequence. It was noted that inactivation of the pbsC gene also affected other iron-regulated phenotypes of Pseudomonas M114.     

Structural and genetic properties of the Eb recombinational hotspot in the mouse

The Eb gene of the mouse contains a recombinational hotspot which plays a predominant role in meiotic crossing-over within the I region of the mouse major histocompatibility complex (MHC). The nucleotide sequences of five recombinants derived from H-2 ^k /H-2 ^b heterozygotes at the Eb locus placed the sites of recombination in each recombinant haplotype within a 2.9 kilobase (kb) segment located fully within the second intron of the Eb gene. Further resolution of the crossover sites was not possible since the nucleotide sequences of the parental and recombinant haplotypes are identical within this segment. The molecular characterization of these five recombinants considered in conjunction with three previously reported intra-Eb recombinants indicates that there are at least two distinct sites of recombination within the Eb recombinational hotspot. In a related study, an examination of the nucleotide sequence of the H-2 ^p allele of the Eb gene revealed a major genetic rearrangement in the 5' half of the intron in this haplotype. A 597 base pair (bp) nucleotide sequence found in the H-2 ^p haplotype is replaced by a 1634 bp segment found in the H-2 ^b and H-2 ^k haplotypes. Sequence analysis of this 1634 bp segment shows strong nucleotide sequence similarity to retroposon long terminal repeat (LTR), env, and pol genes indicating that this segment of the second intron has evolved through retroposon insertion. The location of these retroposon sequences within the 2.9 kb recombination segment defined by the five H-2 ^k /H-2 ^b recombinant haplotypes suggests a possible relationship between these retroviral elements and site-specific recombination within the second intron of the Eb gene. Offprint requests to: H. C. Passmore     

Cytochrome b-deficiency in a mitochondrial muc1muc2 recombinant of Saccharomyces cerevisiae

Summary Cytochrome b-deficient muc1muc2 recombinants of Saccharomyces cerevisiae carrying two specific mitochondrial mucidin-resistant mutations were not able to grow on nonfermentable substrates with the exception of lactate. Crosses of the selected muc1muc2 recombinant strain H331 with different box mutants yielded progenies of mucidin-resistant colonies in a pattern which demonstrates the double mutation origin of the recombinant cells.Only 20% of the wild type cytochrome b content was spectrally detected in the muc1muc2 recombinant, which, however, contained normal amounts of cytochromes c and a when grown under derepressing conditions. The respiratory activity of mitochondria isolated from the muc1muc2 recombinant was limited at the level of cytochrome b and was specifically resistant to high concentrations of mucidin. Electrophoretic analysis of the radioactive products of mitochondrial protein synthesis revealed no differences in apparent molecular weight and amount of radioactivity accumulated in apocytochrome b of the wild and recombinant strains.These results indicate that the accumulation in the same strain of the two mucidin-resistance mutations muc1muc2 results in an altered assembly of the noncovalently attached heme to the doubly mutated form of apocytochrome b.Publication n⁰ 1663 of the Biology Division of the Commission of European Communities. Financial help of the Fonds National de la Recherche Fondamentale Collective is also acknowledged     

The mitochondrial genome of the fission yeast Schizosaccharomyces pombe. 3. Gene mapping in strain EF1 (CBS 356) and analysis of hybrids between the strains EF1 and ade7-50h-

The Schizosaccharomyces pombe strain EF1 (CBS 356) is haploid, prototrophic, respiratory competent, and of homothallic mating type. From restriction enzyme analysis the length of the mitochondrial genome is 17.3 kilobase pairs, which is in good agreement with the value of 17.1 kilobase pairs determined by electron microscopy. The mitochondrial genome of strain EF1 is thus about 2.3 kilobase pairs shorter than that of strain ade7-50h- (about 19.4 kilobase pairs). A restriction map was constructed for 11 enzymes: For most, but not all of them, the pattern is nearly identical to that of strain ade7-50h-. The genes for the large ribosomal RNA, the subunits 1, 2, and 3 of cytochrome c oxidase, subunits 6 and 9 of ATP synthetase, and cytochrome b were localized by hybridization with mitochondrial DNA probes from Saccharomyces cerevisiae. The gene order was found to be the same in both yeast strains. From Southern hybridization of strain ade7-50h- with nick-translated mitochondrial DNA from strain EF1 it is evident that strain EF1 does not possess the intron, which is present in the cytochrome b gene of Schizosaccharomyces pombe strain ade7-50h-. Crosses between strain ade7-50h- and EF1 demonstrate that both the nuclear and the mitochondrial genomes are able to recombine. The mitochondrial genomes of 2 out of 30 independently isolated hybrids between the two strains are described as the result of recombination between the two parental mitochondrial genomes.     

Molecular cloning of alpha-amylase genes fromDrosophila melanogaster. III. An inversion at theAmy locus in an amylase-null strain

Overlapping clones of the structural gene region for alpha-amylase,Amy, were isolated from a lambda EMBL4 library containing genomic DNA fragments from an amylase-null strain ofDrosophila melanogaster. Southern blot analysis and restriction endonuclease mapping of the cloned region indicate that it contains anAmy gene duplication within an inverted repeat sequence as is characteristic of the genomic arrangement for this species. Spacing between the cloned gene copies is similar to that commonly found in other strains. Evidence is presented for the presence of an inversion 4 to 9 kb in length within the clonedAmy region of the null strain. We postulate a causal relationship between the presence of the inversion and the failure of individuals from the null strain to express amylase. A model is proposed that suggests the inversion may have arisen through intramolecular (or sister-strand) recombination mediated by homologous pairing of the inverted repeat sequences at theAmy locus.This research was supported by NIH Grant GM25255.     

Structure and analysis of the mouse Na+/H+ exchanger (NHE1) gene: Homology and conservation of splice sites

The Na⁺/H⁺ exchanger is a widely distributed integral membrane protein that is responsible for pH regulation in mammalian tissues. We have cloned and analyzed the NHE1 isoform of the mouse genomic Na⁺/H⁺exchanger. A clone from a mouse genomic library contained the NHE1 promoter region and the 5-untranslated region. It also contained the first 121 amino acids of the coding region of the Na⁺/H⁺ exchanger. A splice site occurred after amino acid 121, at the same region as in the human NHE1 gene. The deduced amino terminal coding sequence was 76 and 88% identical to the human and rat NHE1 sequences respectively. The 5-untranslated region was highly homologous to that of other species and two minicistrons contained in the human Na⁺/H⁺ exchanger were present in the mouse sequence. The results show that the deduced protein sequence of the mouse NHE1 gene has a high level of homology with other species and that the splice site of the first intron is conserved. These results suggest that the first large intron may play an important role in the NHE1 gene expression.     

Comparative evolution of the mitochondrial cytochrome <Emphasis Type="Italic">b</Emphasis> gene and nuclear <Emphasis Type="Italic">S7</Emphasis> ribosomal protein gene intron 1 in sinipercid fishes and their relatives

The extant sinipercids are a group of freshwater percoid fishes endemic to East Asia. A recent mitochondrial cytochrome b phylogeny of sinipercids has challenged some aspects for their traditional taxonomy and molecular phylogeny, especially for the monophyly of Sinipercidae. In this study, we analyzed mitochondrial cytochrome b and nuclear encoded S7 ribosomal protein gene intron 1 for 10 sinipercid species and 11 related species to compare the phylogenetic signal and nucleotide substitution properties of these two gene sequences. The length of S7 intron 1 ranged from 461 to 719 bp, but alignment was not difficult, and the indels, the proportion of which in the total nucleotides ranged from 3.76 to 45.83%, were phylogenetically informative. Our results indicate that: (1) the relative rate presented by cyt b is five times that of S7 intron 1; (2) the proportion of phylogenetic information is higher in S7 than in cyt b; (3) S7 intron 1 has more base composition bias, but more uniform nucleotide substitution properties; (4) the overall ratio between transitions and transversions in S7 intron 1 is lower than in cyt b. Maximum parsimony and Bayesian analyses of aligned S7 intron 1 and the combined S7 and cyt b dataset resulted in phylogenies that contained the previously identified genera Siniperca and Coreoperca, whereas the monophyly of Coreoperca cannot be corroborated by separate cyt b analysis. The monophyly of Sinipercidae is not supported in separate and combined dataset analyses, although the alternative hypothesis cannot be significantly rejected based on approximately unbiased tests and Shimodaira–Hasegawa tests. Overall, maximum parsimony analyses result in trees with a lack of phylogenetic resolution in deep nodes, and the signal from S7 intron 1 conflicts the cyt b signal in the combined dataset analyses. The reasons for the poor performance of cyt b to S7 intron 1 in the phylogeny are discussed.     

Deficiency in mRNA splicing in a cytochrome c mutant of neurospora crassa: importance of carboxy terminus for import of apocytochrome c into mitochondria.

Molecular cloning and characterization of cytochrome c cDNA clones of Neurospora crassa wild-type (74A) and a cytochrome c-deficient mutant (cyc1-1) are described. Southern blot analysis of genomic DNA indicates that only one cytochrome c gene exists in the N. crassa genome. The cDNA sequence of the wild-type cytochrome c confirmed the previously determined protein sequence. Sequence analysis of the cyc1-1 cDNA for cytochrome c revealed the presence of a larger open reading frame, owing to the presence of an unspliced intron in the 3' end of the coding region. Splicing of this intron is obviously prevented due to the presence of two base exchanges in the highly conserved intron consensus sequences. Consequently, cyc1-1 synthesizes apocytochrome c with an altered carboxy terminus, 19 amino acids longer than the wild-type cytochrome c, with the final 27 amino acids being of an unrelated sequence. This alteration in the carboxy terminus renders the apocytochrome c incompetent for binding to mitochondria and, consequently, import into mitochondria. Thus, unlike other mitochondrial precursor proteins, where it has been demonstrated that the amino terminus alone is sufficient to target the protein to the mitochondria, an intact carboxy terminus is required for efficient import of apocytochrome c into mitochondria. This is independent confirmation for the view that the import pathway of cytochrome c is unique with respect to all other mitochondrial proteins studied to date.     

Degenerated recognition property of a mitochondrial homing enzyme in the unicellular green alga Chlamydomonas smithii

Target sequence cleavage is the essential step for intron invasion into an intronless allele. DNA cleavage at a specific site is performed by an endonuclease, termed a homing enzyme, which is encoded by an open reading frame within the intron. The recognition properties of them have only been analyzed in vitro, using purified, recombinant homing enzyme and various mutated DNA substrates, but it is unclear whether the homing enzyme behaves similarly in vivo. To answer this question, we determined the recognition properties of I-CsmI in vivo. I-CsmI is a homing enzyme encoded by the open reading frame of the alpha-group I-intron, located in the mitochondrial apocytochrome b gene of the green alga Chlamydomonas smithii. The in vivo recognition properties of it were determined as the frequency of intron invasion into a mutated target site. For this purpose, we utilized hybrid diploid cells developed by crossing alpha-intron-plus C. smithii to intron-minus C. reinhardtii containing mutated target sequences. The intron invasion frequency was much higher than the expected from the in vitro cleavage frequency of the respective mutated substrates. Even the substrates that had very little cleavage in the in vitro experiment were efficiently invaded in vivo, and were accompanied by a large degree of coconversion. Considering the ease of the homing enzyme invading into various mutated target sequences, we propose that the principle bottleneck for lateral intron transmission is not the sequence specificity of the homing enzyme, but instead is limited by the rare occurrence of inter-specific cell fusion.