Somatic cell and molecular genetic analysis of various Chinese hamster ovary (CHO) mutant cells defective in sterol-dependent regulation of cholesterol biosynthesis and LDL receptor expression

Wild-type or normal Chinese hamster ovary cells show coordinate regulation of cholesterol biosynthesis and LDL receptor expression in response to sterol in culture medium. In order to identify genes which play an important role in sterol-mediated regulation of of cholesterol metabolism, we selected mutant cell clones which were abnormal in regulating cholesterol metabolism. So far, we have isolated a total of three novel mutants, each one due to a single gene defect. The major focus of my thesis research has been the cloning of these genes.;Using the amphotericin B enrichment procedure, we have isolated several non-leaky mutant CHO cell clones (M4, M19 and M21) which belong to the same genetic complementation group as the mutant M1 cells previously reported from this laboratory (Limanek, J. S., Chin, J., and Chang, T. Y. 1978, PNAS 75:452-456). All of the 4 mutant cell clones were defective in sterol-dependent coordinate regulation of cholesterol biosynthesis and LDL receptor expression. In addition, we have isolated a novel type of 25-hydroxycholesterol resistant mutant CHO cell clone, designated as W1 cell, using M1, M4, M19, or M21 cells as parental cells. A different mutant cell clone was isolated in this laboratory, mutant 25RA cell, which was shown to be resistant to 25-hydroxycholesterol (Chang, T. Y., and Limanek, J. S. 1980 JBC 225:7787-7795). 25RA and W1 cells grow in medium without cholesterol. Unlike wild-type CHO cells, 25RA and W1 cells have elevated level of cholesterol biosynthesis rate and LDL receptor expression when they are grown in medium containing cholesterol. We have found that the mutations in W1 cell and 25RA cell are different. We also found that the M1 and 25RA genes are different, and that a functional M1 gene is required for expression of the 25RA cell phenotype.;When total human genomic DNA was transfected into mutant M19 cells, it gave transfectant cell clones with wild-type cell phenotype. On the other hand, M19 cells acquired the W1 cell phenotype when they were transfected with total W1 genomic DNA. Using gene transfer experiments, we have identified and cloned unique DNA fragments which are closely linked to the human M1 and the W1 genes. Presently, we are using these linked DNA fragments to do a chromosome walk.