I‐NjaI, a nuclear intron‐encoded homing endonuclease from Naegleria, generates a pentanucleotide 3′ cleavage‐overhang within a 19 base‐pair partially symmetric DNA recognition site

Abstract

Different species of the amoebo-flagellate Naegleria harbor optional group I introns in the nuclear ribosomal DNA that contain open reading frames. Intron proteins from Naegleria jamiesoni, Naegleria andersoni, and Naegleria italica (named I-NjaI, I-NanI and I-NitI, respectively) were expressed in Escherichia coli and found to be isoschizomeric homing endonucleases that specifically recognize and cleave intron-lacking homologous alleles of ribosomal DNA. The I-NjaI endonuclease was affinity purified, characterized in more detail, and found to generate five-nucleotide 3 H staggered ends at the intron insertion site which differs from the ends generated by all other known homing endonucleases. The recognition site was delimited and found to cover an < 19 base-pair partially symmetric sequence spanning both the cleavage site and the intron insertion site. The palindromic feature was supported by mutational analysis of the target DNA. All single-site substitutions within the recognition sequence were cleaved by the purified I-NjaI endonuclease, but at different efficiencies. The center of symmetry and cleavage was found to be completely degenerate in specificity, which resembles that of the subclass IIW bacterial restriction enzymes.

The recent transfer of a homing endonuclease gene

“…Target 1 was HindIII-linearized plasmids [pGPR16 or pCPR16 (11) that contain identical inserts] that contain the intron-less rDNA allele (11). Target 2 was the intron-containing rDNA allele.…”

In vivo Expression of a Group I Intron HEG from the Antisense Strand of Didymium Ribosomal DNA

“…The enzymes were heat-inactivated by a 15 min incubation at 80˚C before 0.5 ml of the treated PCR product was subjected to sequencing using the Cycle Sequencing Kit (USB Corporation) and [a-33 P]dCTP (10 mCi/ml, Amersham). I-DirII digested pGPR16 target vector 12,16 was treated with T4 DNA polymerase to create blunt ends, ligated by T4 DNA ligase, and subsequently transformed into E. coli. Plasmids were isolated from colonies growing on LB plates containing ampicillin and the DNA sequence of the ligation junction was determined using automatic sequencing (Big Dye terminator chemistry, Applied Biosystems).…”

Distribution and evolution of variable group-I introns in the small ribosomal subunit of North AtlanticPorphyra(Bangiales, Rhodophyta)

“…Their data (Goddard & Burt, 1999) supported a constant cycle of invasion, degeneration, loss, and finally re-invasion, with each transition occurring roughly every 2 million years. Haugen et al (1999) came to a similar conclusion, based on reports that nuclear homing endonucleases generate doublestrand breaks at intron-lacking rDNA alleles (Johansen et al, 1997;Elde et al, 1999) and are lethal when expressed in yeast (Lin & Vogt, 1998), and that selection occurs against functional forms of homing endonuclease genes. The-pair wise comparison between the different intra-individual intron types in IRE-2, ENG-8, NWY-7, NWY-8 (the 478-bp insertion or not) showed a higher sequence similarity (99.8-100%) in the larger 1506 introns between individuals when compared with shorter intron variants (96.2-100%).…”