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70,000 (hsc70), which are present in unstressed mouse L cells in the absence of detectable hsp68 gene expression. After heat shock, the rapid rate of de novo hsp68 synthesis is a consequence of the new high steady-state concentration of hsp68 mRNA. MATERIALS AND METHODSCell Culture and Heat Shock Conditions. Mouse L cells (WT-4) were grown in alpha Eagle minimal essential medium with 10% fetal calf serum. Drosophila melanogaster Schneider line 2 (SN-2) cells were grown at 25°C in D22 medium containing 2% fetal calf serum. Mouse L cells were heat shocked at 44°C for 10 min, allowed to recover for 2 hr at 37°C, and labeled for 60 min with L-[35S]methionine. Drosophila SN-2 cells were heat shocked for 3 hr at 37°C (6).Blot Analysis of RNA. RNA was extracted from mouse L cells and SN-2 cells with phenol/NaDodSO4 (7), and poly(A)+ RNA was purified (8). Poly(A)+ RNA was electrophoresed through a 1.5% agarose/6% formaldehyde gel (9) and blotted to nitrocellulose (BA85 Schleicher & Schuell) in 1.5 M NaCI/0.15 M trisodium citrate (Cit) (10). The probe was a 2.2-kilobase (kb) Drosophila hsp7Q gene Sal I fragment isolated from the hybrid plasmid 56H8 (11) and subcloned into pBR322 (plasmid pRN301). The Sal I fragment was purified by centrifugation of digested pRN301 through a 5-20% sucrose gradient, and the isolated fragment was labeled with 32p by nick-translation (2, 12). Blots were prehybridized (without glycine) and hybridized as described (13) kb, kilobase(s).

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