Antibodies against a fused gene product identify the protein encoded by a group II yeast mitochondrial intron.
In the mitochondrial genome of Saccharomyces cerevisiae, introns aI1 and aI2 of the gene encoding the COX1 subunit are the only group II introns with open reading frames (ORFs); these can be translated into two homologous proteins, the maturase aI1 and aI2. These proteins are structurally related to viral reverse transcriptases and have been shown genetically to be involved in pre-mRNA splicing and in the deletion of introns from mitochondrial DNA. To identify these mitochondrial proteins and study their properties more directly, we raised antibodies against a part of the intron aI2 ORF translation product. For this purpose, we constructed series of fusion genes, by joining parts of the genes for protein A or lacZ to different portions of the intron aI2. These were expressed in Escherichia coli as hybrid polypeptides, which were used for the production and identification of specific antibodies against the yeast mitochondrial protein. The antibodies recognized the 57 kDa protein (maturase aI2) that accumulates in two yeast mutants deficient in the splicing of aI2. This protein corresponds to the translation product of the 3' part of intron aI2 and accumulates unaltered in the two cis-acting mutants.
Dipartimento di Chimica Biologica dell'Universitá di Padova, Italy.
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Bergantino
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Carignani
D000908:Antibodies, Fungal; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular; D004272:DNA, Mitochondrial / Q000235:genetics*; D005656:Fungal Proteins / Q000032:analysis*; D005800:Genes, Fungal; D007438:Introns; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D012194:RNA-Directed DNA Polymerase / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000032:analysis; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins
D000908:Antibodies, Fungal; D004272:DNA, Mitochondrial; D005656:Fungal Proteins; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; C066564:aI2 protein, S cerevisiae; D012194:RNA-Directed DNA Polymerase