Identification of three highly expressed replacement histone H3 genes of alfalfa

One genomic and six cDNA clones for the replacement histone H3.2 protein of alfalfa (Medicago sativa) were isolated and sequenced. By gene organization they represent 3 distinct genes. PCR methods were used to confirm that only three intron-bearing histone H3.2 genes of this type exist per haploid genome. They co-exist with approximately 56 copies of the previously characterized replication-dependent, intronless histone H3.1 variant gene. Comparison of the relative expression of few constitutive H3.2 genes with the high S phase expression of the abundant cell cycle-dependent H3.1 genes by mRNA levels and protein synthesis measurements revealed that the replacement histone H3.2 genes are very highly expressed. Structural analysis of the genomic replacement H3.2 gene revealed a unique feature. A repeated polypyrimidine sequence motif in the 5' untranslated region of this gene replaces the ubiquitous intron present in all known replacement H3 genes. A hypothesis is presented that this motif and other, non-randomly distributed polypyrimidine sequences in the introns of replacement histone H3 genes of alfalfa and Arabidopsis, may affect nucleosome assembly. Chromatin repression of these replacement genes would be avoided, consistent with the high, constitutive expression of replacement H3 histone genes in plants.