Figure 3.

Detection of evening-enriched mouse liver cDNA fragments. (A) Sau3A-digested cDNA libraries generated from mouse liver mRNA isolated at 8 a.m. (AM) or 8 p.m. (PM) were used for SABRE selection of evening-enriched species, using the PM tester vs. AM driver (4E), or the AM library as both tester and driver (4C). After four selection rounds, two species enriched in the experimental selection (4E) were isolated (A and B); A, cDNA fragment of unknown coding specificity; B, coumarin 7-hydroxylase gene cDNA fragment. Note that species A is distinct from the abundant species in 4C that migrates slightly faster than AM, size in bp of DNA molecular weight markers. (Right) Overexposure of lanes 4E and 4C to better detect species A. (B) Accumulation of coumarin 7-hydroxylase Sau3AI cDNA fragment in rounds of selection by SABRE. Southern blot analysis on PCR products of a.m. and p.m. libraries (AM, PM), and products of experimental (Exp) and control (Control) hybridization rounds (1 to 4, as indicated), using an RNA probe for the 351-bp coumarin 7-hydroxylase cDNA fragment. (C) Detection of coumarin 7-hydroxylase and species A mRNAs by RNase protection analysis of poly(A)⁺ RNA from BALB/c mouse liver isolated at 9 a.m. (9) or 8 p.m. (20). Numbers at right refer to size in nucleotides of protected RNA fragments.