In wild-type (WT) Chinese hamster ovary (CHO) cells, removal of low density lipoprotein (LDL) from growth medium causes a large activity increase in LDL receptor and various cholesterogenic enzymes. Unlike the WT cell, such activity increase does not occur in a specific lipid auxotroph (mutant clone M1). Biochemical and genetic analyses of clone M1 strongly suggest that the observed defect is caused by a single gene mutation, implying that a common genetic factor controlling the coordinate expression of LDL receptor and various cholesterogenic enzymes may exist. Upon transfection of M1 cell with human DNA, we have isolated a putative primary transformant (PT1). Pilot studies indicate that, PT1 cell grows well in lipid-free medium. Unlike WT cell, PT1 cell is quite resistant to cytotoxicity of 25- hydroxycholesterol at 1 ug/ml. PT1 cell possesses elevated LDL receptor activity and elevated rate of sterol synthesis. Morphology of PT1 cell resembles that of an atherosclerotic foam cell. We ask support to perform the following experiments: (1) Synteny analyses between the inserted human M1 gene and the inserted human gene causing LDL receptor/sterol overproduction in M1 cell. Hybrids between M1 cell and human fibroblast will be formed and selected. Standard techniques of somatic cell genetics will be employed to address the question: Is the gene which causes M1 cell to grown in lipid-free medium (referred to as the human M1 gene) and the human gene which causes M1 cell to overproduce LDL receptor and sterol closely linked in the same human chromosome? (2) Isolation and characterization of CHO cell deletion mutants defective at the M1 gene locus and other loci. Well-characterized delection mutants will later serve as important tools for molecular cloning the human M1 gene via DNA mediated gene transfer. (3) Molecular characterizations of CHO cell mutant M1, PT1, and appropriate M1-human fibroblast hybrids using cDNA probes for LDL receptor and various cholesterogenic enzymes. This analysis addresses the question: Does the M1 gene locus play an important role in controlling the coordinate expression of LDL receptor and cholesterogenic enzymes at the transcriptional level? (4) Molecular cloning of the human M1 gene, and sequencing of its full-length cDNA. Using mutant M1 as the recipient, procedures well-documented in literature for cloning genes via DNA mediated gene transfer will be employed to clone the human M1 gene. In addition, the partial or full length cDNA for M1 protein will be engineered into prokaryotic expression vectors to produce fusion proteins, against which antibodies can be raised.
