Genes within genes: independent expression of phage T4 intron open reading frames and the genes in which they reside.

Gott, Jonatha M.; Zeeh, A.; Bell-Pedersen, Deborah; Ehrenman, Karen; Belfort, Marlene; Shub, David A.

doi:10.1101/gad.2.12b.1791

Cited by 70 publications

(56 citation statements)

References 37 publications

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“…I-SceI, the endonuclease encoded by the intron, is expressed from a minor species derived from cleavage at a certain site (54). Endonucleases of T-even phage group I introns are expressed only at later stages of phage infection (14).…”

Section: Discussionmentioning

confidence: 99%

I-PpoI, the Endonuclease Encoded by the Group I Intron PpLSU3, Is Expressed from an RNA Polymerase I Transcript

Lin

Vogt

1998

Molecular and Cellular Biology

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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...

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Section: Discussionmentioning

confidence: 99%

I-PpoI, the Endonuclease Encoded by the Group I Intron PpLSU3, Is Expressed from an RNA Polymerase I Transcript

Lin

Vogt

1998

Molecular and Cellular Biology

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“…To our knowledge, intron-internal promoters for homing endonuclease ORFs have been reported for only one other system, the group I introns of T4 phage (17). The T4 intron ORFs are expressed from these promoters at fairly high levels late in infection and, interestingly, are not expressed from the early pre-mRNAs.…”

Section: Discussionmentioning

confidence: 99%

“…Cr.psbA4 homing was also used to demonstrate exon coconversion and the trans functioning of the intron ORF. Surprisingly, homing of exogenous Cr.psbA4 does not require an external promoter, and expression of the ORF can be driven from a promoter within the intron itself, a phenomenon which heretofore has been reported only for bacteriophage T4 introns (17). The highly efficient homing of exogenous Cr.psbA4 using a DNA fragment that contains little more than the intron may have implications for lateral intron movement and for improving organelle transformation.…”

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confidence: 93%

Mobile Self-Splicing Group I Introns from the psbA Gene of Chlamydomonas reinhardtii: Highly Efficient Homing of an Exogenous Intron Containing Its Own Promoter

Odom

Holloway

Deshpande

et al. 2001

Molecular and Cellular Biology

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Introns 2 and 4 of the psbA gene of Chlamydomonas reinhardtii chloroplasts (Cr.psbA2 and Cr.psbA4, respectively) contain large free-standing open reading frames (ORFs). We used transformation of an intronless-psbA strain (IL) to test whether these introns undergo homing. Each intron, plus short exon sequences, was cloned into a chloroplast expression vector in both orientations and then cotransformed into IL along with a spectinomycin resistance marker (16S rrn). For Cr.psbA2, the sense construct gave nearly 100% cointegration of the intron whereas the antisense construct gave 0%, consistent with homing. For Cr.psbA4, however, both orientations produced highly efficient cointegration of the intron. Efficient cointegration of Cr.psbA4 also occurred when the intron was introduced as a restriction fragment lacking any known promoter. Deletion of most of the ORF, however, abolished cointegration of the intron, consistent with homing. The Cr.psbA4 constructs also contained a 3-(3,4-dichlorophenyl)-1,1-dimethylurea resistance marker in exon 5, which was always present when the intron integrated, thus demonstrating exon coconversion. Remarkably, primary selection for this marker gave >100-fold more transformants (>10,000/g of DNA) than did the spectinomycin resistance marker. A trans homing assay was developed for Cr.psbA4; the ORF-minus intron integrated when the ORF was cotransformed on a separate plasmid. This assay was used to identify an intronic region between bp ؊88 and ؊194 (relative to the ORF) that stimulated homing and contained a possible bacterial (؊10, ؊35)-type promoter. Primer extension analysis detected a transcript that could originate from this promoter. Thus, this mobile, self-splicing intron also contains its own promoter for ORF expression. The implications of these results for horizontal intron transfer and organelle transformation are discussed.Some group I introns are characterized by the ability to self-splice in vitro and to promote their insertion into intronless copies of the host gene in vivo (reviewed in references 10 and 29). The latter process is a type of homologous recombination known as intron homing. Such mobile introns typically contain a large open reading frame (ORF) that codes for a site-specific endonuclease. The endonuclease promotes homing by producing a double-strand break in the intronless allele near the site of intron insertion. A popular model for the subsequent events is based on the double-strand break repair model of DNA recombination in Saccharomyces cerevisiae (36), a postulate of which is that coconversion of flanking exon sequences accompanies intron acquisition.Mobile group I introns have been found in phylogenetically diverse eucaryotes and in certain bacteriophages (29). In eucaryotes, they occur in mitochondrial, chloroplast, and nuclear genes but are typically more common in the organelles. Group I intron homing has been studied intensively, however, only in bacteriophage T4, which has provided an elegant viral-procaryotic system for studying homing mechanism...

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“…Although little is known of the parameters that are required for efficient translation in Synechocystis, it is possible that high expression of I-Ssp6803I might be deleterious even in its natural cellular environment. Interestingly, the RBSs of the homing endonuclease genes in phage T4 are also sequestered in RNA secondary structures that regulate their expression (Gott et al, 1988).…”

Section: Discussionmentioning

confidence: 99%

A novel group I intron-encoded endonuclease specific for the anticodon region of tRNAfMet genes

Bonocora

Shub²

2001

Mol Microbiol

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Genes within genes: independent expression of phage T4 intron open reading frames and the genes in which they reside.

Cited by 70 publications

References 37 publications

I-PpoI, the Endonuclease Encoded by the Group I Intron PpLSU3, Is Expressed from an RNA Polymerase I Transcript

I-PpoI, the Endonuclease Encoded by the Group I Intron PpLSU3, Is Expressed from an RNA Polymerase I Transcript

Mobile Self-Splicing Group I Introns from the psbA Gene of Chlamydomonas reinhardtii: Highly Efficient Homing of an Exogenous Intron Containing Its Own Promoter

A novel group I intron-encoded endonuclease specific for the anticodon region of tRNAfMet genes

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