Sheep Kidney Nuclease

Abstract

An endonuclease showing no apparent base specificity, nor any preference for the sugar moiety has been isolated from sheep kidney. The products of the digestion are 5′‐P‐ended oligonucleotides, and not mononucleotides. Preparation of short oligo A and oligo U (di‐, tri‐, tetra‐) with a good yield is described. The enzyme requires Mg ions, is inhibited at high ionic strength and destroyed by temperatures of 60° and above.The enzyme displays an extremely rigorous specificity with regard to the secondary structure of its substrate. This structural specificity is apparent from the fact that native DNA, complexes between poly A and poly U, and polyinosinic acid in high salt are completely resistant to the enzyme. Even poly A and poly C, at neutral pH denatured DNA, ribosomal RNA, and especially tRNA, are only slowly attacked.Preliminary experiments on the effect of addition of poly U on the hydrolysis of a copolymer A, C suggest that the enzyme will hydrolyze the unstructured loop of polyribonucleotide complexes.The analysis of the hydrolysis of tRNAs by sheep kidney unclease shows that the enzyme attacks all chains and produces mainly large fragments (average chain length, 40 nucleotide units), containing pGp terminal and TpΨpCpG sequences; and small oligonucleotides (average chain length 4.5 nucleotide units), containing a large amount of fragments with a CpCpA terminal.These results are discussed in relation to the conformation of tRNA.