A group I intron in the chloroplast large subunit rRNA gene of Chlamydomonas eugametos encodes a double-strand endonuclease that cleaves the homing site of this intron

Gauthier, Antonin; Turmel, Monique; Lemieux, Claude

doi:10.1007/bf00362086

Cited by 73 publications

(52 citation statements)

References 21 publications

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“…The CeuI sites in the genomic cleavage maps of S. typhimurium LT2 and E. coli K-12 appear to be completely conserved. This is as expected, because the cleavage site of the intron-encoded CeuI enzyme is a 26-bp site in the chloroplast DNA encoding the large-subunit rRNA of C. eugamatos, and this 26-bp site is completely conserved in other chloroplast and mitochondrial DNAs and also in the rrlB gene of E. coli K-12 (12), as well as in many other ribosomal sequences from prokaryotes as determined by a BLAST search of GenBank sequences. This high degree of conservation of sequence is common in ribosomal sequences (56).…”

Section: Fig 3 (A)supporting

confidence: 79%

“…

The 90-kb plasmid pSLT has oneXbaI site and one BinI site.

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mentioning

confidence: 99%

“…CeuI is an endonuclease, derived from a group I intron in Chlamydomonas eugametos, which digests a 26-bp sequence in the gene for the large-subunit rRNA (12,30). We previously reported that CeuI cuts the genome of S. typhimurium into only seven fragments through cleavage of the rRNA (rrn) genes (26).…”

mentioning

confidence: 99%

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The XbaI-BlnI-CeuI genomic cleavage map of Salmonella typhimurium LT2 determined by double digestion, end labelling, and pulsed-field gel electrophoresis

1993

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Endonuclease digestion of the 4,800-kb chromosome of SabloneUa lyphimurium LT2 yielded 24 XbaI fragments, 12 BIn! fragments, and 7 CeuI fragments, which were separated by pulsed-field gel electrophoresis.The 90-kb plasmid pSLT has oneXbaI site and one BinI site. The locations of the fragments around the circular chromosome and of the digestion sites of the different endonucleases with respect to each other were determined by excision of agarose blocks containing fragments from single digestion, redigestion with a second enzyme, end labelling with 32P by using T7 DNA polymerase, reelectrophoresis, and autoradiography. Forty-three cleavage sites were established on the chromosome, and the fragments and cleavage sites were designated in alphabetical order and numerical order, respectively, around the chromosome. One hundred nine independent TnlO insertions previously mapped by genetic means were located by pulsed-field gel electrophoresis on the basis of the presence of XbaI and Bin1 sites in TnlO. The genomic cleavage map was divided into 100 units called centisomes; the endonuclease cleavage sites and the genes defined by the positions of Tn1O insertions were located by centisome around the map. There is very good agreement between the genomic cleavage map, defined in centisomes, and the linkage map, defined in minutes. All seven rRNA genes were located on the map; all have the Ceu! digestion site, all four with the tRNA gene for glutamate have theXbaI and the BinI sites, but only four of the seven have the Bin! site in the 16S rRNA (rrs) gene. Their inferred orientation of transcription is the same as in Escherichia coli. A rearrangement of the rrnB and rrnD genes with respect to the arrangement in E. coli, observed earlier by others, has been confirmed. The sites for all three enzymes in the rrn genes are strongly conserved compared with those in E. coli, but the XbaI and BinI sites outside the mrn genes show very little conservation.

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Section: Fig 3 (A)supporting

confidence: 79%

“…

The 90-kb plasmid pSLT has oneXbaI site and one BinI site.

…”

mentioning

confidence: 99%

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The XbaI-BlnI-CeuI genomic cleavage map of Salmonella typhimurium LT2 determined by double digestion, end labelling, and pulsed-field gel electrophoresis

1993

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“…5) in the chloroplast large subunit rRNA gene (rrnL) of the green alga Chlamydomonas eugametos introduces a double-strand break near the insertion site of the intron in cognate intronless allele (Gauthier et al, 1991 ;Marshall and Lemieux, 1991). In interspecific crosses between C. eugametos and Chlamydomonas moewusii, this double-strand break initiates a very efficient unidirectional gene conversion event (Lemieux and Lee, 1987) during which the intron is most probably inserted by the double-strand break repair mechanism proposed by Szostak et al (1983).…”

mentioning

confidence: 99%

The I‐Ceu I endonuclease: purification and potential role in the evolution of Chlamydomonas group I introns

Marshall

Davis

Lemieux

1994

European Journal of Biochemistry

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During genetic crosses between the interfertile green algae Chlamydomonas eugametos and Chlamydomonas moewusii, the I‐Ceul endonuclease encoded by the fifth group I intron (CeLSU · 5) in the C. eugametos chloroplast large subunit rRNA gene mediates the mobility of this intron by introducing a double‐strand break near the insertion site of the intron in the corresponding C. moewusii intronless allele. To characterize the biochemical properties of this endonuclease, we have purified I‐CeuI and determined the optimal reaction conditions for cleavage. I‐CeuI activity is maximal at 50°C, pH 10.0, 2.5 mM MgCl2 and in the absence of NaCl. Unlike the well‐characterized I‐SceI endonuclease, I‐CeuI remains stable following preincubation in the absence of substrate. We discuss the role that homing endonucleases may have played in the evolution of Chlamydomonas chloroplast group I introns.

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“…Homing endonuclease I-CeuI protein sequence (Gauthier et al, 1991) was back translated into DNA sequence using the BackTranslate program of the GCG Wisconsin Package (Accelrys Inc., Burlington, MA) using maize (Zea mays) highly expressed gene codons except that AGC, CCC, and CTG were used for Ser, Pro, and Leu, respectively. Because expression of I-CeuI endonuclease is toxic to Escherichia coli, a 189-bp potato (Solanum tuberosum) ST-LS1 modified intron sequence (Narasimhulu et al, 1996) was inserted into the I-CeuI endonuclease coding sequence to prevent its expression in bacteria.…”

Section: Construction Of I-ceui Endonuclease Expression Vectorsmentioning

confidence: 99%

Targeted Integration of T-DNA into the Tobacco Genome at Double-Stranded Breaks: New Insights on the Mechanism of T-DNA Integration

2003

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Agrobacterium tumefaciens T-DNA normally integrates into random sites in the plant genome. We have investigated targeting of T-DNA by nonhomologous end joining process to a specific double-stranded break created in the plant genome by I-CeuI endonuclease. Sequencing of genomic DNA/T-DNA junctions in targeted events revealed that genomic DNA at the cleavage sites was usually intact or nearly so, whereas donor T-DNA ends were often resected, sometimes extensively, as is found in random T-DNA inserts. Short filler DNAs were also present in several junctions. When an I-CeuI site was placed in the donor T-DNA, it was often cleaved by I-CeuI endonuclease, leading to precisely truncated targeted T-DNA inserts. Their structure requires that T-DNA cutting occurred before or during integration, indicating that T-DNA is at least partially double stranded before integration is complete. This method of targeting full-length T-DNA with considerable fidelity to a chosen break point in the plant genome may have experimental and practical applications. Our findings suggest that insertion at break points by nonhomologous end joining is one normal mode of entry for T-DNA into the plant genome

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A group I intron in the chloroplast large subunit rRNA gene of Chlamydomonas eugametos encodes a double-strand endonuclease that cleaves the homing site of this intron

Cited by 73 publications

References 21 publications

The XbaI-BlnI-CeuI genomic cleavage map of Salmonella typhimurium LT2 determined by double digestion, end labelling, and pulsed-field gel electrophoresis

The XbaI-BlnI-CeuI genomic cleavage map of Salmonella typhimurium LT2 determined by double digestion, end labelling, and pulsed-field gel electrophoresis

The I‐Ceu I endonuclease: purification and potential role in the evolution of Chlamydomonas group I introns

Targeted Integration of T-DNA into the Tobacco Genome at Double-Stranded Breaks: New Insights on the Mechanism of T-DNA Integration

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