Figure 3.

A. Localization of the U₅H transcripts. CHO cells transfected with the U₅H gene were fractionated into nuclear (N), polysomal (P) and cytosol (S) fractions and equal cell equivalents of each fraction assayed by S1 nuclease mapping using the H2a-614 gene labeled at the 3′ end of the Aval site at codon 20 as a probe as in Figure 2A. The protected fragments are: H2a_H–protection of the endogenous hamster H2a mRNAs; H2a_M–protection of the RNA expressed from the U₅H gene. B. Effect of hydroxyurea on U₅H RNA. Duplicate cultures of CHO cells transfected with the U₅H gene were treated with 5mM hydroxyurea for one hour and RNA prepared from control (lanes 1 and 3) and treated cultures (lanes 2 and 4). 5 μg total cell RNA was assayed by SI nuclease mapping using the U₅H gene labeled at the 5′ end of the Narl site at codon 43 as a probe. Lane M is marker pUC18 digested with HpaII. The protected fragments are: H2a_H–protection to the ATG codon by the endogenous hamster H2a mRNAs; U1–protection to the first nucleotide in the U1 sequence of the U₅H gene.