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...
The activities of viral and insect promoters were examined in a range of insect cell lines permissive and nonpermissive for the replication of the baculovirus Autographa californica nuclear polyhedrosis virus. Recombinant baculoviruses were constructed to place the bacterial chloramphenicol acetyltransferase gene under the control of promoters strongly active in the early, late, or very late stages of virus replication. In fully permissive cells, expression from a very late promoter was 2-to 3-fold higher than expression from a late promoter and 10to 20-fold higher than expression from an early promoter or from a virus-borne insect promoter. In cell lines that do not support the efficient production of viral progeny, late-promoter-driven expression was similar to or surpassed very late promoter-driven expression. In nonpermissive insect cell lines, expression driven by an insect promoter derived from Drosophila melanogaster was higher than expression from the three viral promoters and was especially high in the Drosophila cell line tested. Surprisingly, late-promoter-driven expression, which is dependent on DNA replication, was higher than early-promoterdriven expression in three of four nonpermissive lines. In contrast, very late promoter-driven expression was quite limited in nonpermissive cell lines. The results indicate that the promoter used to drive foreign-gene expression strongly influences the range of insect cells which can efficiently support the production of the foreign protein during infection with recombinant baculoviruses. Three phases of transcriptional activation-early, late, and very late-can be distinguished during the replication cycle of the baculovirus Autographa californica nuclear polyhedrosis virus (AcAMNPV). Early viral transcripts are
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