The mechanisms responsible for the periodic accumulation and decay of histone mRNA in the mammalian cell cycle were investigated in mouse erythroleukemia cells, using a cloned mouse H3 histone gene probe that hybridizes with most or all H3 transcripts. Exponentially growing cells were fractionated into cell cycle-specific stages by centrifugal elutriation, a method for purifying cells at each stage of the cycle without the use of treatments that arrest growth. Measurements of H3 histone mRNA content throughout the cell cycle show that the mRNA accumulates gradually during S phase, achieving its highest value in mid-S phase when DNA synthesis is maximal. The mRNA content then decreases as cells approach G2. These results demonstrate that the periodic synthesis of histones during S phase is due to changes in the steady-state level of histone mRNA. They are consistent with the conventional view in which histone synthesis is regulated coordinately with DNA synthesis in the cell cycle. The periodic accumulation and decay of H3 histone mRNA appear to be controlled primarily by changes in the rate of appearance of newly synthesized mRNA in the cytoplasm, determined by pulse-labeling whole cells with [3HJuridine. Measurements of H3 mRNA turnover by pulse-chase experiments with cells in S and G2 did not provide evidence for changes in the cytoplasmic stability of the mRNA during the period of its decay in late S and G2. Furthermore, transcription measurements carried out by brief pulse-labeling in vivo and by in vitro transcription in isolated nuclei indicate that the rate of H3 gene transcription changes to a much smaller extent than the steady-state levels of the mRNA or the appearance of newly synthesized mRNA in the cytoplasm. The results suggest that post-transcriptional processes make an important contribution to the periodic accumulation and decay of histone mRNA and that these processes may operate within the nucleus.The biosynthesis of histones is generally thought to occur periodically in the cell cycle and to be tightly coupled to DNA replication. Early studies utilizing synchronized HeLa cells showed that newly synthesized histones could be found associated with chromatin only in S-phase cells (2,14,27). These cells contained an abundance of small polyribosomes with nascent polypeptides that comigrated with histones in gel electrophoresis; these small polysomes were not apparent in G1 cells (2). When cells undergoing DNA replication are treated with inhibitors of DNA synthesis there is a rapid loss of histone production (2, 4). Gallwitz and Mueller (8) showed that histone proteins were not synthesized in vitro on microsomes isolated from such cells, and Borun et al. (1) confirmed these results by a direct mRNA translation assay.Despite extensive investigations into the mechanisms responsible for the temporal pattern of histone synthesis in the cell cycle, there is still considerable disagreement concerning the level(s) at which histone protein synthesis is regulated. A number of studies support the view ...

The regulated expression of a mouse histone gene was studied by DNA-mediated gene transfer. A chimeric H3 histone gene was constructed by fusing the 5' and 3' portions of two different mouse H3 histone genes. Transfection of the chimeric gene into mouse fibroblasts resulted in the production of chimeric mRNA at levels nearly equal to that of the total endogenous H3 histone mRNAs. Most chimeric RNA transcripts had correct 5' and 3' termini, and the chimeric mRNA was translated into an H3.1 protein that accumulated in the nucleus of the transfected cells. Expression of the chimeric gene was studied under several conditions in which the rate of transcription and the stability of endogenous H3 transcripts change. Chimeric mRNA levels were regulated in parallel with endogenous H3 mRNAs, suggesting that cis-acting regulatory sequences lie within or near individual histone genes. In addition to correctly initiated and terminated chimeric mRNA, we also detected a novel H3 transcript containing an additional 250 bases at the 3' end. Surprisingly, the longer transcript is polyadenylated and accumulates in the cytoplasm.Histone genes provide an attractive system to study the factors involved in the control of gene expression because they constitute a multigene family whose expression is regulated coordinately during the mitotic cell cycle. The histone genes from several organisms have been isolated and their sequences determined. With some exceptions, histone genes and mRNAs from many organisms share common structural features, including the absence of intervening sequences and the presence of a 3'-terminal stem-loop structure rather than the polyadenylate [poly(A)] tail present on most eucaryotic mRNAs (7,13,14). Recent evidence suggests that the 3' end of histone mRNAs is formed by a posttranscriptional cleavage mechanism in several nonmammalian organisms (2,11,19,24). There are about 20 genes coding for the replication-type H3 histone proteins in mouse cells (9) and probably an equal number for each of the other core histone proteins as well. Graves and co-workers have isolated 10 histone genes, including 4 H3 genes, from three different genomic clusters and have shown that each of the genes is expressed in cultured mouse cells (Sa). Thus, for each type of histone protein there is a family of coordinately regulated mRNAs which contains common coding region sequences. However, these RNAs differ in the 5' and 3' untranslated regions, and the sequences flanking the genes show little homology with one another (13).The level of histone mRNAs in growing cells is regulated periodically with DNA synthesis during the mitotic cell cycle (1,3,6,18). When cells are treated with drugs that inhibit DNA synthesis, the level of histone mRNAs declines rapidly (6, 23). Studies in several laboratories have shown that this coordinate regulation of histone mRNA levels occurs through a combination of transcriptional and posttranscriptional controls (1,3,6

The mechanisms responsible for the periodic accumulation and decay of histone mRNA in the mammalian cell cycle were investigated in mouse erythroleukemia cells, using a cloned mouse H3 histone gene probe that hybridizes with most or all H3 transcripts. Exponentially growing cells were fractionated into cell cycle-specific stages by centrifugal elutriation, a method for purifying cells at each stage of the cycle without the use of treatments that arrest growth. Measurements of H3 histone mRNA content throughout the cell cycle show that the mRNA accumulates gradually during S phase, achieving its highest value in mid-S phase when DNA synthesis is maximal. The mRNA content then decreases as cells approach G2. These results demonstrate that the periodic synthesis of histones during S phase is due to changes in the steady-state level of histone mRNA. They are consistent with the conventional view in which histone synthesis is regulated coordinately with DNA synthesis in the cell cycle. The periodic accumulation and decay of H3 histone mRNA appear to be controlled primarily by changes in the rate of appearance of newly synthesized mRNA in the cytoplasm, determined by pulse-labeling whole cells with [3H]uridine. Measurements of H3 mRNA turnover by pulse-chase experiments with cells in S and G2 did not provide evidence for changes in the cytoplasmic stability of the mRNA during the period of its decay in late S and G2. Furthermore, transcription measurements carried out by brief pulse-labeling in vivo and by in vitro transcription in isolated nuclei indicate that the rate of H3 gene transcription changes to a much smaller extent than the steady-state levels of the mRNA or the appearance of newly synthesized mRNA in the cytoplasm. The results suggest that post-transcriptional processes make an important contribution to the periodic accumulation and decay of histone mRNA and that these processes may operate within the nucleus.

The regulated expression of a mouse histone gene was studied by DNA-mediated gene transfer. A chimeric H3 histone gene was constructed by fusing the 5' and 3' portions of two different mouse H3 histone genes. Transfection of the chimeric gene into mouse fibroblasts resulted in the production of chimeric mRNA at levels nearly equal to that of the total endogenous H3 histone mRNAs. Most chimeric RNA transcripts had correct 5' and 3' termini, and the chimeric mRNA was translated into an H3.1 protein that accumulated in the nucleus of the transfected cells. Expression of the chimeric gene was studied under several conditions in which the rate of transcription and the stability of endogenous H3 transcripts change. Chimeric mRNA levels were regulated in parallel with endogenous H3 mRNAs, suggesting that cis-acting regulatory sequences lie within or near individual histone genes. In addition to correctly initiated and terminated chimeric mRNA, we also detected a novel H3 transcript containing an additional 250 bases at the 3' end. Surprisingly, the longer transcript is polyadenylated and accumulates in the cytoplasm.