IDENTIFICATION OF 3 HIGHLY EXPRESSED REPLACEMENT HISTONE H3 GENES OF ALFALFA

One genomic and six cDNA clones for the replacement histone H3.2 prote in of alfalfa (Medicago sativa) were isolated and sequenced. By gene o rganization 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 hist one H3.1 variant gene. Comparison of the relative expression of few co nstitutive 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 mo tif in the 5' untranslated region of this gene replaces the ubiquitous intron present in all known replacement H3 genes. A hypothesis is pre sented that this motif and other, non-randomly distributed polypyrimid ine sequences in the introns of replacement histone H3 genes of alfalf a and Arabidopsis, may affect nucleosome assembly. Chromatin repressio n of these replacement genes would be avoided, consistent with the hig h, constitutive expression of replacement H3 histone genes in plants.