BACKGROUND:We have previously isolated a thermolabile nuclease specific for double-stranded DNA from industrial processing water of Northern shrimps (Pandalus borealis) and developed an application of the enzyme in removal of contaminating DNA in PCR-related technologies.METHODOLOGY/PRINCIPAL FINDINGS:A 43 kDa nuclease with a high specific activity of hydrolysing linear as well as circular forms of DNA was purified from hepatopancreas of Northern shrimp (Pandalus borealis). The enzyme displayed a substrate preference that was shifted from exclusively double-stranded DNA in the presence of magnesium to also encompass significant activity against single-stranded DNA when calcium was added. No activity against RNA was detected. Although originating from a cold-environment animal, the shrimp DNase has only minor low-temperature activity. Still, the enzyme was irreversibly inactivated by moderate heating with a half-life of 1 min at 65 degrees C. The purified protein was partly sequenced and derived oligonucleotides were used to prime amplification of the encoding cDNA. This cDNA sequence revealed an open reading frame encoding a 404 amino acid protein containing a signal peptide. By sequence similarity the enzyme is predicted to belong to a family of DNA/RNA non-specific nucleases even though this shrimp DNase lacks RNase activity and is highly double-strand specific in some respects. These features are in agreement with those previously established for endonucleases classified as similar to the Kamchatka crab duplex-specific nuclease (Par_DSN). Sequence comparisons and phylogenetic analyses confirmed that the Northern shrimp nuclease resembles the Par_DSN-like nucleases and displays a more distant relationship to the Serratia family of nucleases.CONCLUSIONS/SIGNIFICANCE:The shrimp nuclease contains enzyme activity that may be controlled by temperature or buffer compositions. The double-stranded DNA specificity, as well as the thermolabile feature, strengthens its potential for in vitro applications.

Several species within the amoeboflagellate genus Naegleria harbor an optional ORF containing group I introns in their nuclear small subunit ribosomal DNA. The different ORFs encode homing endonucleases with 65 to 95% identity at the amino-acid level. I-NjaI, I-NanI and I-NitI, from introns in Naegleria jamiesoni, N. andersoni and N. italica, respectively, were analyzed in more detail and found to be isoschizomeric endonucleases that recognize and cleave an approximal 19-bp partially symmetrical sequence, creating a pentanucleotide 3' overhang upon cleavage. The optimal conditions for cleavage activity with respect to temperature, pH, salt and divalent metal ions were investigated. The optimal cleavage temperature for all three endonucleases was found to be 37 degrees C and the activity was dependent on the concentration of NaCl with an optimum at 200 mM. Divalent metal ions, primarily Mg2+, are essential for Naegleria endonuclease activity. Whereas both Mn2+ and Ca2+ could substitute for Mg2+, but with a slower cleavage rate, Zn2+ was unable to support cleavage. Interestingly, the pH dependence of DNA cleavage was found to vary significantly between the I-NitI and I-NjaI/I-NanI endonucleases with optimal pH values at 6.5 and 9, respectively. Site-directed mutagenesis of conserved I-NjaI residues strongly supports the hypothesis that Naegleria homing endonucleases share a similar zinc-binding structure and active site with the His-Cys box homing endonuclease I-PpoI.

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' 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 approximate to 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.

Protists are unicellular eukaryotes which represent a significant fraction of the global biodiversity. The myxomycete Didymium and the schizopyrenid amoeboflagellate Naegleria are distantly related protists. However, we have noted several striking similarities in life cycle, cell morphology, and ribosomal DNA organization between these organisms. Both have multicopy nuclear extrachromosomal ribosomal DNAs. Here the small subunit ribosomal RNA genes are interrupted by an optional group I twintron, a novel category among the group I introns. Group I twintrons are mobile self-splicing introns of 1.3–1.4 kb in size, with a complex organization at the RNA level. A group I twintron consists of two distinct ribozymes (catalytic RNAs) with different functions in RNA processing, and an open reading frame encoding a functional homing endonuclease—all with prospects of application as molecular tools in biotechnology. Updated RNA secondary structure models of group I twintrons, as well as an example of in vitro ribozyme activity, are presented. We suggest that the group I twintrons have been independently established in myxomycetes and schizopyrenid amoeboflagellates by horizontal gene transfer due to a combination of the phagocytotic behavior in natural environments and the extrachromosomal multicopy nature of ribosomal DNA.

Abstract Group I introns are sporadically, but universally distributed among different genes and genomes. They are distinguished from other classes of introns by a characteristic RNA structure and splicing mechanism. Group I introns may encode two different gene products with relevance to biotechnology: group I ribozymes and rare-cutting homing endonucleases. General aspects on molecular and biological features of naturally occurring group I introns are briefly discussed in the first part of the chapter. The second part focuses on group I ribozymes and their potential as tools in molecular biology and therapy, including both gene therapy and chemotherapy. Finally, rare-cutting homing endonucleases are reviewed and considered as molecular tools in genome mapping and genome engineering.

DiSSU1 is an optional group I twintron present in the nuclear extrachromosomal ribosomal DNA of the myxomycete Didymium iridis. DiSSU1 appears to be complex both in structure and function. At the RNA level it has a twin-ribozyme organization composed of two group I ribozymes with different functions, separated by an open reading frame. Here, we show that DiSSU1 is mobile when haploid intron-containing and intron-less amoebae are mated. The mobility process is fast, being completed in 5-10 nuclear cycles after mating in the developing zygote and plasmodia. Analyses of progeny from genetic crosses confirm intron mobility. DiSSU1 is the first example of a mobile group I twintron. The intron-encoded protein was expressed in Escherichia coli and found to be an endonuclease, I-DirI, that cleaves an intron-less ribosomal DNA allele at the intron-insertion site, and is probably involved in intron homing. The endonuclease I-DirI seems to be a rare example of a protein that is expressed from a ribozyme-processed RNA polymerase I transcript in vivo.