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Three enzymes that cause inhibition of mRNA translation, eukaryotic initiation factor 2 protein kinase PK-i, oligoisoadenylate synthetase E, and phosphodiesterase 2'-PDi, have been recently isolated from interferon-treated cells. We show that the rise in these three enzyme activities may be used to study the response of uninfected cells to interferon. (17) formed by a dsRNA-dependent enzyme, the oligoisoadenylate synthetase E, in interferon-treated cells (6, 9,13,18,19 4 ml) or 96-well microtiter plates (2-3 X 104 cells in 0.1 ml per well). One day after seeding, fresh medium with various doses of interferon was added. For kinetic experiments, 200 units of interferon per ml was added so that cells could be harvested simultaneously. Mouse interferon, produced in L cells by infection with Newcastle disease virus, was partially purified to 107 units/mg of protein by successive treatment with acid (pH 2), ammonium sulfate precipitation, and chromatography on CM-cellulose (5). Monkey and human interferon were produced and used on monkey BSC-1 and human FS11 cells as described (25).Preparation of Cell Extracts. The plates were cooled on ice and washed three times with 35 mM Hepes, pH 7.5/140 mM NaCl/3 mM MgCl2. To lyse the cells, 0.2 ml (for 60-mm plates) or 0.025 ml (for microplate wells) of 20 mM Hepes, pH 7.5/5 mM MgCl2/120 mM KCl/1 mM dithiothreitol/10% glycerol (Buffer B) containing 0.5% Nonidet P40 (NP40) was added. After 6-9 min of agitation, the extracts from 60-mm plates were centrifuged for 6 min in an Eppendorf Microfuge (8000 X g).The supernatants (A2Ws about 17 units) could be stored in liquid air, and 2-5 ,ul was sufficient for measuring the various enzymatic activities. For microplates, the whole plate was centrifuged in an International swinging rotor at 3600 rpm for 15 min and 10-15 Mul of the supernatant was used.Assay of PK-i. Aliquots of cell extracts were preincubated in 20 ,ul of Buffer B/0.5 ,ug of poly(rI)-(rC) per ml/0.5 mM ATP for 15 min at 30°C. Then 5 ,tg of eIF-2 and 75 ,M [k-32P]ATP (16 Ci/mmol; 1 Ci = 3.7 X 1010 becquerels) were added and the incubation (25 ul) was continued for 15 min at 300C. Samples were submitted to electrophoresis in polyacrylamide gels in sodium dodecyl sulfate as before (5, 13). The autoradiographs were scanned at 600 nm in a Gilford recording spectrophotometer, and the area under the 35,000-Mr band of eIF-2 was measured in arbitrary units.Abbreviations: PK-i, interferon-induced protein kinase; eIF-2, eukaryotic initiation factor 2; dsRNA, double-stranded RNA; 2'-PDi, interferon-induced phosphodiesterase; NP40, Nonidet P40; VSV, vesicular stomatitis virus. 3208The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

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DNA methylation has been correlated with reduced gene expression in a number of studies, although evidence for a causal link between the two events has been lacking. Because microinjection of simian virus 40 (SV40) DNA into the nucleus of Xenopwu laevis oocytes results in the synthesis of both early and late viral gene products, it was possible to test whether a specific methylation event can affect gene expression. The single SV40 Hpa H site at 0.72 SV40 map units was specifically methylated with Hpa H methylase. When this DNA was injected into oocytes, there was a marked reduction in the synthesis of the major late viral capsid protein VP-1, relative to the synthesis by an unmethylated control. However, production of the early proteins (the large and small tumor antigens) was not affected by Hpa II methylation. Therefore, methylation at a single site on the viral DNA located near the 5' end ofthe late region can specifically repress late gene expression. The possible mechanisms by which this repression is mediated are discussed.The only modified base in eukaryotic DNA is 5-methylcytosine (for review, see refs. 1 and 2). This modification occurs in the DNA of a wide variety of species, from bacteria through vertebrates and plants, which suggests that it may function in gene regulation. The major 5-methylcytosine residue in eukaryotic DNA is located in the dinucleotide sequence C-G. Restriction enzymes Hpa II and Msp I both cleave the sequence C-C-G-G. However, the former cleaves the sequence only if the C-G is unmethylated whereas the latter will cut the sequence in either state. By using the Southern blotting procedure to compare the restriction patterns of DNA cleaved with one or the other enzyme, it is possible to locate methylated C-C-G-G sites in genomic DNA. In this way, a strong correlation between gene activity and the state of DNA methylation has been demonstrated in various systems. For example, tissue-specific differences in the methylation patterns of chicken (3) and human (4) globin, ovalbumin (5), and viral (6, 7) DNA have been observed which suggest that actively expressed genes are under-methylated. This is supported by the observation that actively transcribing chromatin is under-methylated relative to methylated transcriptionally inactive chromatin (8).The experiments in this study were further prompted by the strikingly nonrandom fashion in which C-G residues are arranged on the simian virus 40 (SV40) genome (Fig. 1). Nineteen of the 27 C-G residues are located between 0.66 and 0.75 map units, a region comprising <10% of the viral genome. This region delineates the sequences that form the nucleosome-free "gap" region that has been identified in SV40 minichromosomes (9-11) and contains the majority of the known regulatory sequences of the viral genome including the origin of replication (reviewed in ref. 12), the early (13, 14) and late (15, 16) region promoters, and the 72-base-pair repeat sequence (17) that enhances transcription. In contrast, in the remaining 90% of the genome, whi...

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