The nucleotide sequences of two chicken histone genes encoding replacement variant H3.3 polypeptides are described. Unlike the replication variant genes of chickens (and almost all other organisms), these genes contain intervening sequences; introns are present in both genes in the 5' noncoding and coding sequences. Furthermore, the replacement variant histone mRNAs are post-transcriptionally polyadenylated. The locations, but not the sizes, of the two introns within the coding segments of the two genes have been exactly conserved, whereas the intron positions in their respective 5' flanking regions differ. Although both H3.3 genes predict the identical histone polypeptide sequence, they are as different from one another as each of them is from a more common replication variant H3.2 gene in silent base substitutions within the coding sequences. Thus, the H3.3 polypeptide sequence has been precisely maintained over a great evolutionary period, suggesting that this class of histones performs a strongly selected biological function. Although replacement variant histones can account for more than 50% of the total H3 protein in the nuclei of specific chicken tissues, the steady-state level of H3.3 mRNA is nearly the same (and is quite low) in all tissues and ages of animals examined. These properties suggest novel mechanisms for the control of the basal histone biosynthesis which takes place outside of the S phase of the cell cyde.The molecular architecture imposed upon eucaryotic DNA is principally mediated by the evolutionarily highly conserved family of histone proteins. The histones must elaborate (in concert with less abundant nonhistone chromosomal proteins) the characteristics of primary, secondary, and higher-order packaging of DNA into its highly condensed form within the nucleus.With (12,19,21,37 MATERIALS AND METHODSRecombinant DNA. Histone-encoding lambda genomic recombinants were isolated from a Charon 4A library (8) by using heterologous sea urchin H3 and H2a histone gene probes as previously described (12,42). DNA sequencing and subcloning of relevant segments of lambda CH4a, CH5d, and CH6b (see Fig. 1) was performed as previously described (7). RNA blots, primer extension, and S1 analyses were performed as previously described (7,14,15,27), with the exception that the RNA blot hybridized to the SP6-radiolabeled probe (see Fig. 4) was washed in a final solution of 0.3 M NaCI-10 mM Tris-hydrochloride-5 mM EDTA (final pH, 7.5) containing 4 ,ug of RNase A per ml at 30°C for 1 h.