REGULATION OF ENDOGENOUS AND TRANSFECTED HISTONE GENES IN MAMMALIAN CELLS (CYCLE, MOLECULAR BIOLOGY, TRANSFER)

Abstract

The histones are a group of small basic proteins found in the eukaryotic cell nucleus. They appear to be synthesized only during periods of DNA replication. 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. Exponentially growing cells were fractionated into cell cycle specific stages by centrifugal elutriation. My results demonstrate that the periodic synthesis of histone proteins during S phase is due to changes in the steady state levels of histone mRNA which are controlled primarily by changes in the rate of appearance of newly synthesized mRNA in the cytoplasm, determined by pulse-labeling whole cells with ('3)H-uridine. Changes in histone mRNA half-life do not significantly account for differences in the steady state level of H3 mRNA. 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. These results suggest that post-transcriptional processes make an important contribution to the periodic accumulation of histone mRNA and that these processes may operate within the nucleus.;I undertook to develop a system whereby the control mechanisms governing histone gene regulation can ultimately be dissected at the gene sequence level. A mouse hybrid H3 histone gene (constructed in vitro) was introduced into mouse L cells. Expression of the transfected H3 gene was detected at levels nearly equal to the expression of endogenous H3 histone genes and was regulated in parallel with the expression of the endogenous H3 histone genes when the rate of DNA synthesis was altered. I have generated several transfectant cell lines in which the expression of a transfected histone gene can be regulated similarly to endogenous histone genes. This system can now be exploited to help determine the role of neighboring DNA sequences in the regulation of histone gene expression.