A new specific DNA endonuclease activity in yeast mitochondria

Summary Two group I intron-encoded proteins from the yeast mitochondrial genome have already been shown to have a specific DNA endonuclease activity. This activity mediates intron insertion by cleaving the DNA sequence corresponding to the splice junction of an intronless strain. We have discovered in mitochondrial extracts from the yeast strain 777-3A a new DNA endonuclease activity which cleaves the fused exon A3-exon A4 junction sequence of the COXI gene.     

I-BasI and I-HmuI: two phage intron-encoded endonucleases with homologous DNA recognition sequences but distinct DNA specificities

I-HmuI and I-BasI are two highly similar nicking DNA endonucleases, which are each encoded by a group I intron inserted into homologous sites within the DNA polymerase genes of Bacillus phages SPO1 and Bastille, respectively. Here, we present a comparison of the DNA specificities and cleavage activities of these enconucleases with homologous target sites. I-BasI has properties that are typical of homing endonucleases, nicking the intron-minus polymerase genes in either host genome, three nucleotides downstream of the intron insertion site. In contrast, I-HmuI nicks both the intron-plus and intron-minus site in its own host genome, but does not act on the target from Bastille phage. Although the enzymes have distinct DNA substrate specificities, both bind to an identical 25bp region of their respective intron-minus DNA polymerase genes surrounding the intron insertion site. The endonucleases appear to interact with the DNA substrates in the downstream exon 2 in a similar manner. However, whereas I-HmuI is known to make its only base-specific contacts within this exon region, structural modeling analyses predict that I-BasI might make specific base contacts both upstream and downstream of the site of intron insertion. The predicted requirement for base-specific contacts in exon 1 for cleavage by I-BasI was confirmed experimentally. This explains the difference in substrate specificities between the two enzymes, including the observation that the former enzyme is relatively insensitive to the presence of an intron upstream of exon 2. These differences are likely a consequence of divergent evolutionary constraints.     

The mitochondrial genome of the fission yeast Schizosaccharomyces pombe: highly homologous introns are inserted at the same position of the otherwise less conserved cox1 genes in Schizosaccharomyces pombe and Aspergillus nidulans.

The DNA sequence of the second intron in the mitochondrial gene for subunit 1 of cytochrome oxidase (cox1), and the 3'' part of the structural gene have been determined in Schizosaccharomyces pombe. Comparing the presumptive amino acid sequence of the 3'' regions of the cox1 genes in fungi reveals similarly large evolutionary distances between Aspergillus nidulans, Saccharomyces cerevisiae and S. pombe. The comparison of exon sequences also reveals a stretch of only low homology and of general size variation among the fungal and mammalian genes, close to the 3'' ends of the cox1 genes. The second intron in the cox1 gene of S. pombe contains an open reading frame, which is contiguous with the upstream exon and displays all characteristics common to class I introns. Three findings suggest a recent horizontal gene transfer of this intron from an Aspergillus type fungus to S. pombe. (i) The intron is inserted at exactly the same position of the cox1 gene, where an intron is also found in A. nidulans. (ii) Both introns contain the highest amino acid homology between the intronic unassigned reading frames of all fungi identified so far (70% identity over a stretch of 253 amino acids). However, in the most homologous region, a GC-rich sequence is inserted in the A. nidulans intron, flanked by two direct repeats of 5 bp. The 37-bp insert plus 5 bp of direct repeat amounts to an extra 42 bp in the A. nidulans intron. (iii) TGA codons are the preferred tryptophan codons compared with TGG in all mitochondrial protein coding sequences of fungi and mammalia.(ABSTRACT TRUNCATED AT 250 WORDS)     

Sequence of the intron and flanking exons of the mitochondrial 21S rRNA gene of yeast strains having different alleles at the omega and rib-1 loci

The complete nucleotide sequence has been determined for the intron, its junctions and the flanking exon regions of the 21S rRNA gene in three genetically characterized strains differing by their omega alleles (omega+, omega- and omega n) and by their chloramphenicol-resistant mutations at the rib-1 locus. Comparison of these DNA sequences shows that: --omega+ differs from omega- and omega n by the presence of the intron (1143 bp), as well as by a second and unexpected mini-insert (66 bp) located 156 bp upstream within the exon, whose nature and functions are still unknown but whose striking palindromic structure may suggest a mitochondrial transposable element. --The two mutations C321R and C323R correspond to two different monosubstitutions, 56 bp apart in the omega- and omega n strains but separated by the intron in the omega+ strains. In relation to previous genetic results, a model is discussed assuming that the interactions of two different regions or genetic loci determine the chloramphenicol resistance, one of which contains the omega n mutations. --A long uninterrupted coding sequence able to specify a 235 amino acid polypeptide exists within the intron. This remarkable observation gives new insight into the origin of the mitochondrial introns and raises the question of the possible functions of intron-encoded polypeptides. Finally, sequence comparisons with evolutionarily distant organisms, showing that different rRNA introns are inserted at different positions of an otherwise highly conserved region of the gene, suggest a recent insertion of these introns and a mechanism for splicing after the assembly of the large ribosomal subunit.     

A nuclear gene encoding the beta subunit of the mitochondrial ATP synthase in Nicotiana plumbaginifolia.

The beta subunit of the mitochondrial ATP synthase in Nicotiana plumbaginifolia is encoded by two nuclear genes, atp2-1 and atp2-2, which are both expressed. The complete nucleotide sequence of atp2-1 has been determined. It contains eight introns ranging from 88 to 1453 bp. The last intron contains a putative insertion element (Inp), of 812 bp bordered by 35-bp inverted repeats which share an 11-bp homology with Agrobacterium tumefaciens T-DNA borders. Sequences homologous to Inp are present in multiple copies in the N. plumbaginifolia and the N. tabacum genome but not in more distant species. The atp2-1 encoded polypeptide is highly homologous to beta subunits from other ATP synthases but it contains an extension at the N terminus which is probably involved in mitochondrial targeting. A sequence homology between exon 4 of atp2-1 and exon 1 of the human ras genes suggests a common ancestral origin for these exons.     

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.     

Linkage map of two HLA-SB beta and two HLA-SB alpha-related genes: an intron in one of the SB beta genes contains a processed pseudogene

Three overlapping cosmid clones contain coding sequences for four HLA Class II genes, provisionally identified as two HLA-SB alpha and two HLA-SB beta genes. The genes are in the order beta, alpha, beta, alpha, inverted with respect to each other. One of the SB beta genes contains a 513 bp sequence that appears to be a processed pseudogene, flanked by direct 17 bp repeat sequences, in the intron upstream of the beta 1 exon. The pseudogene is homologous to a family of sequences of approximately 25-40 members, most of which are not on chromosome 6. A cDNA clone, highly homologous to the pseudogene, except for its 5' end, contains a normal poly(A) addition site and a poly(A) tail. The cDNA clone is homologous to a single-copy gene in both man and mouse, encoded on human chromosome 15. A search of published DNA sequences identified a mouse sequence, with about 77% similarity to the pseudogene sequence, in the negative strand of an intron in a mouse dihydrofolate reductase gene. The second SB beta gene does not contain the pseudogene sequence.     

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.     

Complete structure of the hamster alpha A crystallin gene. Reflection of an evolutionary history by means of exon shuffling

The eye lens contains a structural protein, alpha crystallin, composed of two homologous primary gene products alpha A2 and alpha B2. In certain rodents, still another alpha crystallin polypeptide, alpha AIns, occurs, which is identical to alpha A2 except that it contains an insertion peptide between residues 63 and 64. In this paper we describe the complete alpha A crystallin gene that has been cloned from DNA isolated from Syrian golden hamster. Evidence is provided that the alpha A gene is present as a single copy in the hamster genome. The detailed organization of the gene has been established by means of DNA sequence analysis and S1 nuclease mapping, revealing that the gene consists of four exons. The first exon contains the information for the 68 base-pair long 5' non-coding region as well as the coding information for the first 63 amino acids. The second exon encodes the 23 amino acid insertion sequence, the third exon codes for amino acid 87 to 127 of the alpha AIns chain, whereas the last exon encodes the C-terminal 69 amino acids and contains the information for the 523 base-pair long 3' non-coding region. The second exon is bordered by a 3' splice junction (A X G/G X C), which deviates from the consensus for donor splice sites (A X G/G X T). This deviation is found in both hamster and mouse. An internal duplication was detected in the first exon by using a DIAGON-generated matrix for comparison. By means of similar DIAGON-generated matrices it was confirmed that the amino acids coded for by the third and fourth exons are homologous to the small heat-shock proteins of Drosophila, Caenorhabditis and soyabean. The implications of the differential splicing and the evolutionary aspects of the detected homologies are discussed.     

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.     

The structure of the gene encoding chain c of the hemoglobin of the earthworm, Lumbricus terrestris

The complete nucleotide sequence of the gene for chain c of hemoglobin of the earthworm Lumbricus terrestris has been determined. The sequence of 4037 base pairs (bp) includes about 310 bp of 5'-flanking sequence and 110 bp 3' to the poly(A) site. Comparison of cDNA and genomic sequences shows four silent differences in codons that suggest the presence of at least two genes. The coding sequence is split by two introns of 1344 and 1169 bp at highly conserved positions (Jhiang, S. M., Garey, J. R., and Riggs, A. F. (1988) Science 240, 334-336). The first intron possesses the unusual 5' splice junction sequence GC instead of GT. Many tandem triplet repeats based on (GAT) and (CCT) are present in the first intron. The second intron has nine tandem repeats based on the consensus sequence AAGGAAGGAGGTC. Each intron has several exact inverted repeats of 9-10 bp that might result in loops of 78-140 nucleotides in the RNA prior to splicing. The sequences in the second intron, at positions 2423-2644 are about 65% identical with parts of several genes found in yeast mitochondria and in DNA from several other organisms.