Pheromone 3 mRNA of the ciliate Euplotes octocarihatus contains three in-frame UGA codons that are translated as cysteines. This was revealed from cDNA sequencing and from plasma desorption mass spectrometry of cleaved pheromone 3 in connection with pyridylethylation of the fragments. N-terminal sequence analysis of carboxymethylated protein confirmed this conclusion for the first of the three UGA codons. Besides UGA the common cysteine codons UGU and UGC are also used to encode cysteine. UAA functions as a termination codon. Preparation of RNA. Total RNA was prepared by disruption of 1-3 x 107 cells in 8 M urea/4 M LiCl in a PotterElvehjem homogenizer, followed by precipitation on ice overnight. RNA was collected by centrifugation, dissolved in 10 mM Mops, pH 7.5/0.5% SDS, and extracted three times with phenol/chloroform/isoamyl alcohol (25:24:1) and once with chloroform/isoamyl alcohol (24:1). Total RNA was then precipitated by addition of 0.1 volume of 4 M LiCl and 2.5 volumes of absolute ethanol.Poly(A)+ RNA was prepared by affinity chromatography on oligo(dT)-cellulose (Bethesda Research Laboratories) as recommended by the supplier with the exception that Mops was used as the buffer instead of Tris. Poly(A)+ RNA was precipitated by the addition of LiCI and ethanol as described above and redissolved in water. Quantity and quality were determined spectrophotometrically by measuring absorption at 260 and 280 nm (14).cDNA Synthesis and Cloning. The cDNA library was constructed (15) in the vector AgtlO. The cDNA was treated with S1 nuclease and ligated with EcoRI linkers prior to its insertion into the EcoRI site of the vector. The pheromone 3 gene was identified by plaque hybridization with the synthetic oligodeoxynucleotide 5'-GTRTANGGYTCYTCCCA-3', corresponding to the N terminus of the secreted pheromone, and was isolated by standard techniques (14).DNA Sequencing. Eight positively hybridizing plaques were obtained from 105 transformants. Five of them were further subcloned for sequencing by the dideoxy chain-termination method. Their nucleotide sequences were determined from double-stranded and single-stranded templates (pUC12, pT7T3, M13mpl8, and M13mp19 as sequencing vectors) according to the sequencing strategy outlined in Fig. 1 1To whom reprint requests should be addressed.

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The effects of binary combinations of the recombinant human cytokines, interleukin-lp (rHuIL-lP), tumor necrosis factor a (rHuTNFa), and y-interferon (rHuyIFN) on the production of prostaglandin E (PGE), hyaluronic acid (HA), and collagenase by human synovial fibroblasts in culture were investigated. All 3 were stimulated by rHuIL-l/? and rHuTNFa alone, but not by rHuy-IFN. Stimulation with rHuIL-lP and rHuTNFa occurred at femtomolar and picomolar concentrations, respectively, and maximal stimulation by rHuIL-lP was several times greater than that by rHuTNFa. Stimulation of PGE and collagenase production with rHuIL-lp or rHuTNFa was depressed by rHuyIFN, depending on the concentration used. In contrast, stimulation of HA production with rHuIL-lP or rHuTNFa was unaffected or increased somewhat with rHuy-IFN. Combinations of rHuIL-lP and rHuTNFa had marked synergistic effects on PGE and collagenase production. However, when rHuIL-1P effects were maximal, rHuTNFa had an additive effect. These cytokines had only additive effects on HA produc-

Recombinant human cytokines were compared for their effects on glycosaminoglycan (GAG) synthesis in human synovial fibroblast cultures and human articular cartilage explant cultures. In fibroblast cultures, recombinant human interleukin-la (rHuIL-la), rHuIL-lP, and recombinant human tumor necrosis factor a (rHuTNFa) stimulated hyaluronic acid (HA) production and, to a lesser extent, sulfated GAG production, while recombinant human y-interferon did not have a significant effect. Half-maximal stimulation of HA by rHuIL-lP was 0.14 pM, while stimulation for rHuIL-la and rHuTNFa was 1.6 pM and 32 pM, respectively. Indomethacin (10 pg/ml) had no influence on HA stimulation by cytokines, while hydrocortisone ( 2 1 0 pg/ml) caused a significant reduction. In articular cartilage cultures, the cytokines inhibited production of