The proliferation of animal cells proceeds through a tightly regulated series of events termed the mitotic cell cycle. Recent observations linking cell cycle regulated genes, growth factors, and oncogenes suggest that an understanding of the molecular processes responsible for normal cell cycle regulation may also prove to be an important step in the identification of the molecular events associated with the loss of proliferation control in cancer cells. A biochemical characterization of the transcriptional control mechanisms utilized by the cell cycle regulated gene dihydrofolate reductase will provide a model system for understanding growth regulation. The murine dihydrofolate reductase gene is cell cycle regulated at the transcriptional level, resulting in a transient increase in transcription rate at the G1/S boundary. Preliminary experiments suggest that this increased transcription is modulated by a transcription factor(s) whose specific activity changes as cells progress through the cell cycle. Identification and purification of this cell cycle-specific transcription factor(s) will be carried out through two complementary approaches. Hybridization of radiolabeled DNA fragments containing the dhfr promoter region to in vitro transcription extract immobilized on nitrocellulose filters will detect specific DNA-protein interactions. Examination of the binding properties of stage-specific extracts will not only determine if the cell cycle regulation of the dhfr gene is directed by a DNA binding protein, but will simultaneously allow an assignment of molecular weight to the transcription factor(s). Protein- protein interactions involved in the regulation of the dhfr gene will be examined by addition of fractionated S phase extracts to isolated transcription complexes formed using non-S phase extracts. Stage-specific proteins which bind to the core transcription complex will be detected by enhanced in vitro transcription. Cell lines which are potential overproducers of this transcription factor(s) will also be used as a source for analysis and purification. Once the applicability of this system has been demonstrated with the dhfr promoter, the analysis will be extended to other cell cycle regulated genes. The question can then be addressed as to whether a general mechanism confers cell cycle regulation on a group of genes or whether each gene is independently controlled by its own transcription factors. The existence of common control mechanisms is suggested by similar promoter sequences recently discovered in other growth regulated genes and oncogenes. The ultimate goal is to obtain genomic clones of these regulatory proteins. Peptide analysis of the purified proteins will be used to generate oligonucleotide probes to screen genomic libraries. in addition, stage-specific cDNA libraries will be constructed to identify transcription factors as well as other genes regulated by these factors. Once the genomic clones are obtained, in vivo manipulation of these cell cycle regulatory proteins will then be possible, with the purpose of understanding how they influence the growth properties of normal and abnormal cells.
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