Studies of X-chromosome inactivation with an improved histochemical technique for ornithine carbamoyltransferase

Studies of X-linked enzymes provide an approach to the study of tumour and normal cellular development. We have assessed the technique for the histochemical demonstration of one such enzyme, ornithine carbamoyltransferase (EC 2.1.3.3). Various stages in the Mizutani technique for ornithine carbamoyltransferase were re-examined, and the resulting improved technique applied to normal mice and to mice of the sparse fur strain (Spf) known to have an abnormal form of ornithine carbamoyltransferase inherited as an X-linked characteristic. Positive enzyme activity was present in all hepatocytes from normal mice, the strongest reaction being present in the periportal area with a gradual reduction of activity towards the centrilobular region. No activity was demonstrable in hepatocytes from hemizygous male Spf mice. In heterozygous female Spf mice, there was a clear-cut separation of ornithine carbamoyl-transferase-positive and -negative cells. These were present in very variable proportions in different liver lobes and different animals. Preliminary studies were also carried out using a high pH reaction mixture to detect the abnormal enzyme. These studies demonstrate conclusively the X-linkage of ornithine carbamoyltransferase in mice, showing the mosaic pattern of distribution predicted by the Lyon hypothesis. They show that the Spf strain of mice can be used for studies of both development and tumorigenesis in the liver, and that histochemical study of an animal strain with an X-linked enzyme abnormality provides a powerful investigative tool.