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The human multiple drug resistance (MDR) gene has been used as a selectable marker to increase the proportion of bone marrow cells that contain and express this gene by drug selection. By constructing retroviral vectors containing and expressing the MDR gene and a nonselectable gene such as the f-globin gene, enrichment for cells contining both of these genes can be achieved. A retroviral construct containing MDR cDNA in a Harvey virus-based vector has been used to transfect our ecotropic 3T3 retroviral packagin line GP+E86. Clones have been isolated by exposure of the retrovirally transfected cells (MDR producer cells) to colchicine (60 ng/ml), a selective agent that kills MDR-negative cells. Flow cytometry analysis (fluorescence-activated cell sorting) with an antibody to MDR demonstrates expression of human MDR protein on the surface of these colchicine-resistant producer clones. Untransfected GP+E86 cells are negative. Colchicineresistant clones were titered using clone supernatants and the highest titer clone (4 x 104 viral particles per ml) was cocultured with 10' donor mouse bone marrow cells for 24-48 hr.The donor cells were then injected into congenic irradiated mice, and the presence of the MDR gene was assayed by the polymerase chain reaction (PCR) analysis using MDR-specifIc primers. In one experiment eight of nine transduced mice were positive for MDR by PCR of peripheral blood 14 and 50 days posttransplantation; after 240 days three of nine transduced mice were positive. Bone marrow obtained from one of these positive animals was stained with the MDR monoclonal antibody and the granulocyte population was analyzed by FACS.

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The major excreted protein (MEP) purified from Kirsten-virus-transformed 3T3 fibroblasts and mature human cathepsin L were compared in respect to a number of catalytic criteria and found to be similar. The Mr of MEP is 39,000, whereas that of mature human cathepsin L is 30,000. Sequence data suggested that MEP could be a pro-form of mouse cathepsin L. Both enzymes acted on the synthetic substrate benzyloxycarbonyl-Phe-Arg-7-(4-methyl)coumarylamide with similar catalytic constants and acted optimally at pH 5.5. Both were rapidly inactivated by the active-site-directed inhibitors benzyloxycarbonyl-Phe-Phe-diazomethane and L-3-carboxy-trans-2,3-epoxypropionyl-leucylamido-(4-guanidin o)butane, and furthermore, 3H-labelled L-3-carboxy-trans-2,3-epoxypropionyl-leucylamido-(4-acetamid o)butane, which binds covalently to the heavy chain of mature cathepsin L, also bound to MEP. MEP autolyses rapidly at pH 3.0 to give lower-Mr (35,000 and 30,000) forms, but all forms react with the radiolabelled inhibitor. No autolysis occurred above pH 5.0. MEP hydrolysed azocasein at pH 5.0, demonstrating that it is capable of hydrolysing protein substrates without autolytic activation. Unlike mature forms of cathepsin L, MEP is stable, but not active, at neutral pH. The present work shows that cathepsin L can be secreted as a higher-Mr precursor that is stable in extracellular fluids but only active where local pH values fall below 6.0. These results suggest that the extra N-terminal peptide on MEP is not an activation peptide, but is a regulatory peptide affecting the pH-stability and activity of mouse cathepsin L.