Molecular Cloning of cDNA Encoding Xenopus laevis Deoxyribonuclease I

Abstract

A 1200-bp cDNA encoding Xenopus laevis deoxyribonuclease I (X. laevis DNase I) was constructed from the total RNA of a X. laevis pancreas using a rapid amplification of cDNA ends method. When the cDNA was transiently transfected into COS-7 cells, the recom-binant polypeptide exhibited similar enzymological properties to those of the native pancreatic DNase I. The recombinant enzyme was considerably more labile than most other vertebrate DNase I enzymes. The X. laevis DNase I polypeptide was larger than any other known vertebrate DNase I, containing a unique Cys-rich stretch of 68 or 70 amino acid residues at the carboxyl terminus, and it had less well conserved binding sites for the Ca²⁺, G-actin and DNA, and two DNase I signature motifs. These alterations might account for its heat instability.

Key Words:

• cDNA
• deoxyribonuclease I
• molecular cloning
• transfection
• Xenopus laevis