Cloning and characterisation of the cDNA and gene for sheep liver arginase : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University, Palmerston North

Abstract

Arginase (arginine amidinohydrolase, EC 3.5.3.1) is a ubiquitous enzyme, notably found in the liver of ureotelic animals. It plays a critical role in the hepatic metabolism of most higher organisms as a cardinal component of the urea cycle (Jenkinson et al., 1996). Arginase has also been identified in numerous organisms and tissues where there is no functioning urea cycle. In animals, many extrahepatic tissues have been shown to contain a second form of arginase. closely related to the hepatic enzyme but encoded by a distinct gene or genes and involved in a host of physiological roles. Recent interest in arginase has been stimulated by it's demonstrated involvement with the metabolism of nitric oxide. Subcloning the sheep hepatic cDNA sequence would allow a ruminant arginase to be compared with other known arginases. Probing a sheep genomic library for the arginase gene could ultimately lead to the characterisation of regulatory elements of the gene. Partial purification of sheep liver arginase was carried out to develop a DNA probe to screen a sheep liver cDNA library for the cDNA sequence but the protein was N-terminally blocked. An attempt was made to electroelute arginase from an SDS-PAGE gel with a view to cleaving the purified protein and sequencing some of the resulting peptides. But arginase could not be purified sufficiently for successful electoelution. Total RNA was isolated from both sheep and rat liver. A product of the expected size was produced by RT-PCR on the rat RNA template, but could not be subcloned into a vector. PCR performed on a sheep cDNA library generated a PCR product which was subcloned and sequenced. The sequence had no similarity with known arginase sequences, and showed that the reverse primer sequence was present at both ends of the PCR product. A region of the human arginase cDNA sequence was PCR amplified from the expression plasmid pTAA12. The PCR product was radiolabelled, and used as a probe to screen a sheep liver cDNA library. No positive clones were identified. Northern blot analysis of RNA isolated from sheep liver was carried out. The blot was probed with a fragment of the human arginase cDNA sequence. Nonspecific binding was observed.