PpLSU3, a mobile group I intron in the rRNA genes of Physarum polycephalum, also can home into yeast chromosomal ribosomal DNA (rDNA) (D. E. Muscarella and V. M. Vogt, Mol. Cell. Biol. 13:1023-1033, 1993). By integrating PpLSU3 into the rDNA copies of a yeast strain temperature sensitive for RNA polymerase I, we have shown that the I-PpoI homing endonuclease encoded by PpLSU3 is expressed from an RNA polymerase I transcript. We have also developed a method to integrate mutant forms of PpLSU3 as well as the Tetrahymena intron TtLSU1 into rDNA, by expressing I-PpoI in trans. Analysis of I-PpoI expression levels in these mutants, along with subcellular fractionation of intron RNA, strongly suggests that the full-length excised intron RNA, but not RNAs that are further cleaved, serves as or gives rise to the mRNA.Group I introns are a class of RNA elements that share a secondary structure which allows the intron to undergo selfsplicing from the primary transcript (5). While most group I introns are located in the genes of mitochondria and chloroplasts of lower eucaryotes, some are found in nuclear genes. Interestingly, nuclear group I introns reside only in rDNA, the gene encoding rRNA, and when present they occupy all of the ca. 200 rDNA copies typical of eucaryotic organisms. Some group I introns are mobile genetic elements. They encode a site-specific endonuclease that recognizes and cleaves a DNA sequence at or near the intron insertion site of the intronlacking allele. The double-strand break is then repaired by replication of the intron into the intron-lacking allele, thus converting all intron-lacking alleles into intron-containing alleles. This process is termed intron homing due to its high specificity (3).Among the ca. 150 nuclear group I introns reported so far, only three have been shown or have been inferred to be mobile: DiSSU1 from the slime mold Didymium iridis (6, 21, 22), NaSSU1 from the protist Naegleria andersoni and other Naegleria species (8), and PpLSU3 from the slime mold Physarum polycephalum. Originally found in the large-subunit rDNA gene of the Carolina strain (29), PpLSU3 contains the open reading frame (ORF) for the homing endonuclease I-PpoI (for nomenclature of intron-encoded endonucleases, see reference 7) in its 5Ј half and the ribozyme part in its 3Ј half. The sequence of the ribozyme part of PpLSU3 is 70% identical to the Tetrahymena thermophila intron TtLSU1, which is inserted at the same location as PpLSU3, suggesting a common evolutionary origin. Previous work has shown that PpLSU3 RNA not only undergoes self-splicing but also cleaves itself at an internal processing site (IPS), thus separating the I-PpoI ORF and the ribozyme (38). I-PpoI recognizes a 13-to 15-bp DNA sequence in a portion of the large-subunit rRNA gene that is 100% identical in all eucaryotes (9, 52). When a plasmid-borne PpLSU3 is transformed into Saccharomyces cerevisiae, most cells die upon the induction of I-PpoI expression, because I-PpoI makes double-strand breaks in the ca. 120 rDNA repeats on chromosome...
PpLSU3, a mobile group I intron found in the ribo-somal RNA genes of Physarum polycephalum, encodes the I-PpoI homing endonuclease. This enzyme represents one of the rare cases in nature where a protein is expressed from an RNA polymerase I transcript. Our previous results showed that the full length intron, but not a further processed species, is the messenger for I-PpoI, implying a role of the untranslated region (UTR) in gene expression. To study the function of the 3'-UTR in expression of the endonuclease and in splicing of the intron, we replaced the I-PpoI gene in PpLSU3 with the gene for the alpha-fragment of Escherichia coli beta-galactosidase, and then integrated this chimeric intron into all the chromosomal rDNA repeats of yeast. The resulting cells synthesized functional alpha-fragment, as evidenced by a complementation assay analogous to that used in E.coli. The beta-galactosidase activity thus provides an unusual and potentially valuable readout for Pol I transcription from chromosomal rDNA. This is the first example in which a eucaryotic homing endonuclease gene has been successfully replaced by a heterologous gene. Using deletion mutagenesis and a novel randomization approach with the alpha-fragment as a reporter, we found that a small segment of the 3'-UTR dramatically influences both splicing and protein expression.
We have determined the sequence of an autonomously-replicating circular DNA element from mitochondria of strain T40 of the maize pathogen Cochliobolus heterostrophus (1), a filamentous Ascomycete. The sequence was determined by the dideoxy method, using subclones in M13mplO and mpll. Sequence analysis with Microgenie (Beckman) revealed the presence of the complete gene encoding subunit 6 of the mitochondrial ATPase complex. The ATPase 6 ORF extends between bases 307 and 1080 of the sequence presented below, which is numbered starting from the first base of the unique BamHI site. We have published additional analysis of this plasmid-like DNA elsewhere (2).
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