Molecular analysis of the histone gene cluster of Psammechinus miliaris: III. Polarity and asymmetry of the histone-coding sequences.

Fragments of histone DNA produced by restriction endonucleases contain 5' and 3' termini with defined topologies relative to the histone-coding sequences. After limited resection with lambda-exonuclease, the 6 kb Hindlll histone DNA fragment (see Schaffner et al., 1976) hybridizes to H4 histone mRNA whether or not the DNA has been denatured. This shown that the coding sequence for the histone H4 is proximal to the 3' terminus of the Hindlll restriction fragment. By contrast, lambda-exonuclease digestion of the 6 kb EcoRI histone DNA fragment drastically reduces hybridization of the H4 mRNA. Hence in this molecule, the H4 DNA sequence is near a 5' terminus of the EcoRI restriction fragment. From these results and those described in the preceding paper (Schaffner et al., 1976), the polarity of the H4 gene is therefore 5' H2B leads to H4 leads to H1 3'. Cloned Psammechinus histone DNA (S. G. Clarkson, H. Smith, W. Schaffner, K. Gross, and M. Birnstiel, manuscript submitted for publication) may be strand-separated by electrophoresis. Highly purified histone mRNAs (Gross et al., 1976) all hybridize almost exclusively to the strand of lesser electrophoretic mobility. It follows that all coding sequences are arranged in tandem within the same DNA strand, and that hence they share the same polarity as the H4 DNA sequences. Transcription therefore proceeds in the gene cluster in the direction H4 leads to H2B leads to H3 leads to H2A leads to H1.