Sequence of the 5′-end of Strongylocentrotus purpuratus H2b histone mRNA and its location within histone DNA

Abstract

EXTENSIVE DNA sequence analysis of the cloned histone gene repeat unit from the sea urchin Stronglyocentrotus purpuratus has identified the five histone protein coding sequences^1–3 (I.S., unpublished). R-loop and heteroduplex mapping strongly suggest that the sea urchin histone genes contain no intervening sequences⁴. In addition to the protein coding sequences the corresponding histone mRNAs each contain about 100 extra nucleotides^4,5. The location of these additional sequences in relation to the coding sequences is not certain. It is of interest to map the mRNA termini within the known DNA sequence because this information may help to identify transcriptional and translational control elements. Direct nucleotide sequence analysis of RNA can be carried out by enzymatic, base-specific cleavage of end-labelled RNAs^6,7. However, 5′-end labelling of mRNAs involves several enzymatic reactions (‘decapping’, dephosphorylation and phosphorylation) that require nuclease-free enzyme preparations⁸; furthermore, the RNA to be sequenced must be homogeneous. Indirect sequencing analysis of RNA can be obtained from its complementary cDNA. RNA has been sequenced in this way using a chain termination method with dideoxytriphosphates⁹, synthetic primers and purified mRNA templates^10–12. Alternatively, end-labelled restriction fragments have been used to prime synthesis of cDNA on homogeneous ^13,14 and partially purified^15,16 mRNA templates followed by either chemical¹⁷ or enzymatic⁹ sequencing of the resulting cDNA. Our approach is similar and shows that prior purification of the RNA template is unnecessary. We present here the 5′-terminal sequence of S. purpuratus H2b mRNA, derived from purified H2b mRNA as well as from total polysomal RNA, using the chain termination method⁹ adapted for RNA sequencing. We also determine its location within the histone DNA sequence.