Identification of regulatory sequences in the prelude sequences of an H2A histone gene by the study of specific deletion mutants in vivo

Abstract

Conserved DNA sequence elements of putative regulatory functions were deleted from the prelude region of a sea urchin H2A histone gene. For this, the wild-type H2A gene of the 6-kilobase histone DNA repeat unit was replaced by various mutant H2A genes by cloning. The effects of the manipulation on H2A mRNA synthesis were studied by injection of the mutant DNAs into centrifuged Xenopus oocytes. The unmanipulated H2B gene residing within the same repeat unit provided a suitable internal control for these studies. Deletion of the T-A-T-A-A-A-T-A motif, once thought to be the functional equivalent of the bacterial Pribnow box, did not abolish transcription of the gene; instead, a number of novel mRNA 5' termini were generated. We argue that the T-A-T-A-A-A-T-A motif is a specificity element, a selector of eukaryotic gene transcription. Deletion of the "cap-sequence," 5' pyrimidine-C-A-T-T-C-purine 3' and most of the mRNA leader sequence did not abolish transcription but created yet another mRNA 5' terminus. In contrast to these deletions, which are both down-mutations, deletion of H2A gene-specific conserved DNA sequences upstream from the T-A-T-A-A-A-T-A motif enhanced mRNA synthesis. A hypothesis for the function of these DNA sequences as eukaryotic promoter elements is discussed.