DNA OF NORMAL AND MALIGNANT CELLS

We will measure the temporal order of replication in several cell lines of at least 30 different segments of the mouse genome containing several structural genes including members of the globin and immunoglobulin multigene families and the genes for alphafeto-protein, albumin, actin, myosin, ribosomal RNA and dihydrofolate reductase. We will determine the temporal order of replication of these genes in different cell lines in which they are active or inactive. The information obtained will help clarify the apparent relationship between late DNA replication and genetic inactivity. Criteria for activity in these cells will be the amount of mRNA sequences present and the accessibility of the genes when nuclei are incubated with endonucleases. We have determined that an adult alpha-1 globin region replicates early during S in a subcloned Friend murine erythroleukemia (MEL) cell line. We also have evidence that two beta-globin gene regions replicate during a defined S phase interval in this cell line. The temporal replication of these as well as several other genes will be measured in different murine cell lines to determine the stringency with which this regulation is maintained. This will provide a basis for further biochemical studies on the regulation of activation of specific replicons in mammalian cells. The variable and constant region genes of the immunoglobulin light and heavy chain gene clusters will be examined in detail to determine whether related genes are replicated during the same interval of the S phase. Initial studies will focus on determining whether the temporal replication of any immunoglobulin genes occurs during an interval different from the other members of the gene cluster. Several methods will be used to look for the presence of origins of replication in well characterized (50-250 kb) regions of the mouse genome. The relative rates of appearance in the cytoplasm during the cell cycle in MEL cells will be measured for mRNAs transcribed from genes whose temporal replications we have determined.