Cellular levels of H1 and core histone mRNAs have been examined in exponentially growing HeLa S3 cells as a function of DNA synthesis inhibition under varying concentrations of three DNA synthesis inhibitors. Total cellular histone mRNAs were analyzed by Northern blot hybridization, and their relative abundance was shown to be stoichiometrically and temporally coupled to the rate of DNA synthesis. In the presence of cytosine arabinoside, hydroxyurea, or aphidicolin, a rapid, proportionate decrease of histone mRNA levels resulted in an apparent mRNA half-life of less than 10 min. Using inhibitors of transcription and translation, we show that transcription is not necessary for the coordinate decrease of histone mRNA levels that occurs when DNA synthesis is inhibited. When protein synthesis is inhibited by addition of cycloheximide, core and H1 histone mRNAs do not decrease in parallel with reduced rates of DNA synthesis but instead are stabilized and accumulate with time, thus uncoupling histone mRNA levels and DNA replication. These last observations suggest that protein synthesis, either of histones or of some unidentified regulatory molecules, is required for the stoichiometric turnover of H1 and core histone mRNAs coordinate with reduced rates of DNA synthesis.

We describe the isolation and initial characterization of seven independent A Charon 4A recombinant phages which contain human histone genomic sequences (designated AHHG). Restriction maps of these clones and localization of the genes coding for histones H2A, H2B, H3, and H4 are presented. The presence of histone encoding regions in the AHHG clones was demonstrated by several independent criteria including hybridization with specific DNA probes, hybrid selection/in vitro translation, and hybridization of AHHG DNAs to reverse Southern blots containing cytoplasmic RNAs from G1-, S-, and arabinofuranosylcytosine (cytosine arabinoside)-treated S-phase cells. In addition, the AHHG DNAs were shown to protect in vivo labeled H4 mRNAs from S1 nuclease digestion. Based on the analysis of the AHHG clones, human histone genes appear to be clustered in the genome. However, gene clusters do not seem to be present in identical tandem repeats. The AHHG clones described in this report fall into at least three distinct types of arrangement. One of these arrangements contains two coding regions for each of the histones H3 and H4. This RNA is not similar in size to known histone-encoding RNAs and is present in the cytoplasm ofHeLa cells predominantly in the G1 phase of the cell cycle.Histone proteins complex with DNA to form structures, known as nucleosomes, that are fundamental components in the organization of the eukaryotic genome. The synthesis of histone proteins is tightly coupled to DNA replication in a number of higher eukaryotic cells (1-4). Although more definitive experiments are required, several lines ofevidence point toward transcriptional level control of histone gene expression during the cell cycle in HeLa cells and in normal human diploid fibroblasts (5-10). To elucidate the levels and mechanisms of human histone gene regulation, homologous probes specific for individual histone genes, as well as knowledge about the structure and organization of the human histone genes, are required. Cloned human genomic histone sequences should provide such specific probes and are requisite for the identification of those DNA sequences involved in the regulation of these genes. Histone genes of several other species have been cloned and characterized. In sea urchins and Drosophila melanogaster these genes are clustered and tandemly repeated (reviewed in ref. 11), whereas in yeast (12), mouse (13,14), and chicken (15, 16) the histone genes are also clustered but have no apparent repeat.In this paper, we report the isolation and initial character- (19). Phage DNA was isolated by a modification of the method described by Blattner (18). All experiments involving viable bacteriophage and bacteria containing recombinant DNA were performed under conditions specified by the National Institutes of Health guidelines for research involving recombinant DNA.Gene-Specific Histone Probes. Cloned genomic chicken sequences containing H3 and H4 encoding regions were used to screen the human recombinant library. Gene localization in the ...

Two lines of evidence derived from fusion gene constructs indicate that sequences residing in the 5'-nontranslated region of a cell cycle-dependent human H3 histone mRNA are involved in the selective destabilization that occurs when DNA synthesis is terminated. The experimental approach was to construct chimeric genes in which fragments of the mRNA coding regions of the H3 histone gene were fused with fragments of genes not expressed in a cell cycle-dependent manner. After transfection in HeLa S3 cells with the recombinant plasmids, levels of fusion mRNAs were determined by S1 nuclease analysis prior to and following DNA synthesis inhibition. When the first 20 nucleotides of an H3 histone mRNA leader were replaced with 89 nucleotides of the leader from a Drosophila heat-shock (hsp70) mRNA, the fusion transcript remained stable during inhibition of DNA synthesis, in contrast to the rapid destabilization of the endogenous histone mRNA in these cells. In a reciprocal experiment, a histone-globin fusion gene was constructed that produced a transcript with the initial 20 nucleotides of the H3 histone mRNA substituted for the human f3-globin mRNA leader. In HeLa cells treated with inhibitors of DNA synthesis and/or protein synthesis, cellular levels of this histone-globin fusion mRNA appeared to be regulated in a manner similar to endogenous histone mRNA levels. These results suggest that the first 20 nucleotides of the leader are sufficient to couple histone mRNA stability with DNA replication.The human histone genes are a moderately repeated gene family that encode the major structural proteins ofchromatin. It has been well established that histone gene expression and DNA replication are temporally and functionally coupled. The synthesis of most histone proteins (1-6) and the steadystate levels of histone mRNAs (7-12) are closely correlated with DNA synthesis in the S phase of the cell cycle. At the natural end of S phase or following inhibitor-induced termination of DNA synthesis, there is a coordinate and stoichiometric decrease in histone mRNA levels and histone protein synthesis (8)(9)(10)(11)(12)(13). The rapid loss of histone mRNA under these conditions is in contrast to minimal changes in nonhistone mRNA levels. The selective destabilization of histone mRNA during DNA synthesis inhibition is posttranscriptionally mediated; destabilization is not dependent on transcription (11) but requires protein synthesis (11-15). The cellular and molecular basis for histone mRNA turnover, however, remains unresolved.To address molecular mechanisms operative in the selective destabilization of histone mRNAs, we are attempting to identify regions of a cloned, cell cycle-dependent human H3 histone gene (16,17) that are involved in the destabilization of its transcripts. Our approach is to construct fusion genes, in which fragments ofthe mRNA coding regions ofthe cloned human H3 histone gene are fused with fragments of other genes not expressed in a cell cycle-dependent manner. After transfection into HeLa S3 cells, le...

An H1 histone gene was isolated from a 15-kilobase human DNA genomic sequence. The presence of H2A, H2B, H3, and H4 genes in this same 15-kilobase fragment indicates that mammalian core and H1 histone genes are clustered.