Figure 1.
Purified Northern shrimp nuclease and its DNA substrates.
The enzyme after SDS-PAGE and silver-staining (A), its hydrolyzing activity on different DNA substrates (B), and ligation of its digestion products (C) as analyzed by agarose gel electrophoresis. Activities of two preparations of the shrimp dsDNase (1a and 1b) and of a shrimp ss nuclease (2) on a dsDNA PCR product, a heat denatured PCR product (ds+ssDNA) and on supercoiled plasmid DNA are shown. M defines the lane of protein MW standards and of a 100 bp DNA ladder in the relevant gel types, and lanes C represent the respective untreated DNA substrates used. Lane R shows the ligated reaction products from dsDNase digested plasmid DNA.
Figure 2.
Residual activity of purified Northern shrimp nuclease using the modified Kunitz assay after heating at 65°C for indicated periods of time.
Figure 3.
Influence of divalent cations on enzyme activity.
Relative activity of native (A) and recombinant (B) shrimp nuclease in presence of various divalent cations. Enzyme activity was measured using the modified Kunitz assay, and one hundred percent activity was set at 10 mM MgCl2.
Figure 4.
Nuclease activity and specificity against ss- and dsDNA.
Relative activity of Northern shrimp nuclease on double stranded (ds) and heat denatured (ss) DNA as a function of divalent cations (A: magnesium alone, B: magnesium plus calsium) using the modified Kunitz assay. One hundred percent activity was set using dsDNA substrate at 5 mM MgCl2. The ss nuclease-activity represents the relative activity on heat-denatured when compared to native ds calf thymus DNA. C displays relative activities of bovine pancreatic DNase I and P. borealis dsDNase on ssDNA in the described assay buffer or in a conventional Taq-buffer.
Table 1.
Relative activity of various DNases on single- or double-stranded DNA oligonucleotides.
Figure 5.
Examination of RNase activity in the shrimp nuclease preparation.
Total RNA was incubated with 0.2 or 2 units of enzyme in the presence of only MgCl2 or of MgCl2 + CaCl2. Negative control (N: no enzyme), positive control (P: commercial RNase A).
Figure 6.
cDNA and amino acid sequence of Northern shrimp dsDNase.
Coding sequence is shown in lower letter cases with the derived protein sequence below. A predicted protein signal sequence is highlighted in italics. Location of native protein fragments sequences and their sequence identities compared to cDNA-derived amino acid residues are highlighted and underlined, respectively. Primers used for amplification reactions are indicated above their corresponding cDNA sequence. (GenBank accession no. FN554584).
Figure 7.
Multisequence alignment of selected NUC endonucleases.
Retrived sequences were stripped for signal peptide sequences and aligned using Clustal W2. Amino acids are shaded by degree of conservation (grey for major, black for total). An asterix marks the site of a histidine residue previously reported essential for catalytic activity. Recognised endonuclease NS and NUC domains are indicated according to their occurrence in the P. borealis enzyme. The sequences used are from Northern shrimp (PANBO - C9YSL6), Kuruma prawn (PENJP - Q9U5L8), Red king crab (PARCM - B6ZLK3), Fiddler crab (UCACR - Q0GIJ2), Southern house mosquito (CULQU - B0WUW1), and the gram-negative bacterium Serratia marcescens (SERMA–P13717).