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Purification of the dihydrofolate reductases overproduced by 16 independently-derived antifolate-resistant sublines of Chinese hamster lung cells and their drug-sensitive parental cell line, DC-3F, has confirmed our original observation that two molecular weight classes of this enzyme, viz. Mr = 21,000 can be overproduced by drug-resistant cells (Melera, P. W., Wolgemuth, D., Biedler, J. L., and Hession, C. (1980) J. Biol. Chem. 255, 319-322). Of the 16 drug-resistant sublines analyzed, 12 overproduce the 21,000-dalton enzyme, while 4 overproduce the 20,000-dalton enzyme. Both molecular weight classes are found in the drug-sensitive parental line. Two-dimensional polyacrylamide gel electrophoresis of the two molecular weight classes of enzyme has shown that each is composed of two isoelectric forms, and that the relative distributions of the isoelectric forms within a molecular weight class are maintained during overproduction. In vitro translation experiments have demonstrated that the synthesis of these isoelectric forms and their relative distributions are mediated by mRNA. Finally, evidence is presented for the existence of two sets of three dihydrofolate reductase mRNAs in overproducing sublines. Both sets, within a subline, encode the same molecular weight class of dihydrofolate reductase, while each set directs the synthesis of only one of the isoelectric forms within that class. Although the genetic basis for these observations is not yet understood, it is clear that the production of dihydrofolate reductase by Chinese hamster lung cells is complex and probably involves the expression of multiple dihydrofolate reductase genes and/or alleles.

We have purified a human non-pancreatic phospholipase A2 that is present in platelets and is enriched in rheumatoid synovial fluid. The enzyme is calcium-dependent, has a pH optimum of 8-10, and shows a striking preference for substrate presented in the form of Escherichia coli membranes. In the E. coli phospholipase A2 assay the phospholipase exhibits an apparent specific activity of 300 mumol/mg/min. Using oligonucleotide probes based on amino-terminal sequence data, we cloned the corresponding human gene from a genomic DNA library and expressed the gene in animal cells. The protein was secreted from the cells in an active form. The deduced amino acid sequence of the human protein consists of 124 amino acids, contains structural features common to all known phospholipase A2s, and has a half-cystine pattern that is characteristic of the snake venom group II enzymes.

We have cloned a previously undescribed adhesion molecule, VCAM-1, which is induced by cytokines on human endothelial cells and binds lymphocytes. Using a novel method requiring neither monoclonal antibodies nor purified protein, VCAM-1-expressing clones were selected by adhesion to human lymphoid cell lines. VCAM-1 mRNA is present in endothelial cells at 2 hr after treatment with IL-1 or TNF-alpha and is maintained for at least 72 hr; leukocyte binding activity parallels mRNA induction. Cells transfected with VCAM-1 bind the human leukemia lines Jurkat, Ramos, Raji, HL60, and THP1, but not peripheral blood neutrophils. VCAM-1, which belongs to the immunoglobulin gene super-family, may be central to recruitment of mononuclear leukocytes into inflammatory sites in vivo.