Purification and characterization of the<i>in vitro</i>activity of I-<i>Sce</i>I, a novel and highly specific endonuclease encoded by a group I intron

Monteilhet, Claude; Perrin, Arnaud; Thierry, Agnès; Colleaux, Laurence; Dujon, Bernard

doi:10.1093/nar/18.6.1407

Cited by 168 publications

(139 citation statements)

References 18 publications

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“…3 , I-CeuI activity was found to be maximal at 50°C, pH 10.0, 2.5 mM MgCI,, in the absence of NaCl. These conditions are similar to those described for I-SceI (Montheilet et al, 1990). Although some I-CeuI activity can be detected at physiological conditions (25"C, pH 7.5), the observed value is much lower than that obtained under optimal conditions.…”

Section: Optimal Cleavage Conditions and K supporting

confidence: 85%

“…Reaction mixtures contained 0-10 mM MgC12, 0-100 mM NaCl, 2 mM 2-mercaptoethanol, and were buffered with either Tris (pH 7.5-8.5) or diethanolamine (pH 9.0-11 .O) at a final concentration of 10 mM. Cleavage efficiencies were measured by densitometry as described by Montheilet et al (1990). K,,, values of I-CeuI were determined at pH 10.0, 50°C, 2.5 mM MgCI, and at pH 8.5, 37"C, 2.5 mM MgCl,, using approximately l o n g purified I-CeuI in each assay and the ScaI-linarized pBHS plasmid (20-400 nM) as substrate.…”

Section: In Vitro I-ceui Endonuclease Activitymentioning

confidence: 99%

“…These four factors are known to affect the activity of homing endonucleases (Montheilet et al, 1990, Wernette et al, 1990. As shown in Fig.…”

Section: Optimal Cleavage Conditions and K mentioning

confidence: 99%

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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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Section: Optimal Cleavage Conditions and K supporting

confidence: 85%

Section: In Vitro I-ceui Endonuclease Activitymentioning

confidence: 99%

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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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“…To meet this goal, we used the very rare restriction site, I-SceI, for the intron homing endonuclease (Monteilhet et al 1990) and constructed pBAC/oriV/SceI by cloning the NotIless oriV fragment (see legend to Fig. 1 and Methods section) into the PolIk-blunted XhoI site of pBeloBAC11/SceI, provided by the F.R.…”

Section: Resultsmentioning

confidence: 99%

“…1) by cloning the oriV-containing fragment into the XhoI-digested pTrueBlue-BAC2 plasmid (1999 Catalogue of Genomics One). To obtain the TruBlue-BAC2/oriV/SceI vector (pJW419), a 0.5-kb EcoRI-SalI fragment containing the recognition sequence for I-SceI was prepared from pSCM522 (Monteilhet et al 1990), blunted with PolIk, gel-purified, and ligated to EcoN47III-digested and dephosphorylated pTrueBlue-BAC2/oriV (JW406), resulting in pTrueBlue-BAC2/oriV/ SceI (JW419). To construct pIndigoBAC/oriV, a commercially available linearized plasmid, pIndigoBAC-5 (HindIII-Cloning Ready from Epicentre Technologies), was phosphorylated and religated to reconstruct circular pIndigoBAC-5.…”

Section: Construction Of Pbac/oriv Derivativesmentioning

confidence: 99%

Conditionally Amplifiable BACs: Switching From Single-Copy to High-Copy Vectors and Genomic Clones

Wild¹,

Hradečná²,

Szybalski³

2002

Genome Res.

167

145

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The widely used, very-low-copy BAC (bacterial artificial chromosome) vectors are the mainstay of present genomic research. The principal advantage of BACs is the high stability of inserted clones, but an important disadvantage is the low yield of DNA, both for vectors alone and when carrying genomic inserts. We describe here a novel class of single-copy/high-copy (SC/HC) pBAC/oriV vectors that retain all the advantages of low-copy BAC vectors, but are endowed with a conditional and tightly controlled oriV/TrfA amplification system that allows: (1) a yield of ∼ 100 copies of the vector per host cell when conditionally induced with L-arabinose, and (2) analogous DNA amplification (only upon induction and with copy number depending on the insert size) of pBAC/oriV clones carrying >100-kb inserts. Amplifiable clones and libraries facilitate high-throughput DNA sequencing and other applications requiring HC plasmid DNA. To turn on DNA amplification, which is driven by the oriV origin of replication, we used copy-up mutations in the gene trfA whose expression was very tightly controlled by the araC-P araBAD promoter/regulator system. This system is inducible by L-arabinose, and could be further regulated by glucose and fucose. Amplification of DNA upon induction with L-arabinose and its modulation by glucose are robust and reliable. Furthermore, we discovered that addition of 0.2% D-glucose to the growth medium helped toward the objective of obtaining a real SC state for all BAC systems, thus enhancing the stability of their maintenance, which became equivalent to cloning into the host chromosome.

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Functional analysis ofHO gene in delayed homothallism inSaccharomyces cerevisiae wy2

Ekino

Kwon

Goto

et al. 1999

Yeast

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Saccharomyces cerevisiae wy2 exhibits a novel life cycle, with delayed homothallism caused by a defective HO gene. In this strain, gradual diploidization occurs during successive subcultures. Three amino acids of wy2 HO were different from those of wild‐type (wt) HO, which included a nonsense mutation (TAG) from Trp‐292 and two amino acid changes of His‐475 to Leu and Glu‐530 to Lys. The ho gene of heterothallic strain CG379 was also sequenced in this study. Four amino acids of ho were different from those of HO. Among different amino acids in wy2 HO and ho, the alteration of His‐475 to Leu was common between them. His‐475 in HO was previously suggested to be involved in the DNA binding. We constructed a variety of chimeric HO genes by exchanging the corresponding restriction fragments generated from the wt HO, wy2 HO and ho genes. These results and the site‐directed mutagenesis studies allowed us to draw the following conclusions: (a) Gly‐223 is essential for HO activity; (b) mutation of His‐475 to Leu significantly reduces the HO activity; (c) amber mutation (TAG) in wy2 HO can be suppressed inefficiently. Copyright © 1999 John Wiley & Sons, Ltd.

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Purification and characterization of thein vitroactivity of I-SceI, a novel and highly specific endonuclease encoded by a group I intron

Cited by 168 publications

References 18 publications

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

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

Conditionally Amplifiable BACs: Switching From Single-Copy to High-Copy Vectors and Genomic Clones

Functional analysis ofHO gene in delayed homothallism inSaccharomyces cerevisiae wy2

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