Determining the strategy that cells use to adjust DNA replication to different growth rates once again utilizes measurement of the numbers of gene copies. Consider two cell types, one with a doubling time of 1 hour and one with a doubling time of 2 hours. If each cell type requires the full doubling time to replicate its chromosome, then the distributions of structures of replicating chromosomes extracted from random populations of cells growing at the two different rates would be identical (Fig. 1). However, if both cell types replicate their chromosomes in 1 hour, then the cells with the 1-hour doubling time will possess a different distribution of chromosome structures than the cells with the 2-hour doubling time (Fig2). The problem, once again, is that of counting copies of genes. Measuring the number of copies of genes located near the origin and of genes located near the terminus of replication permits these two possible DNA replication schemes to be distinguished.

Fig1. States of chromosomes of cells with 1-hour and 2-hour doubling times assuming 1 hour or 2 hours are required to replicate the DNA. The ratio of copies of genes near the replication origin and replication terminus are the same for cells growing with the two rates.

Fig2. States of chromosomes in cells with a 2-hour doubling time, assuming only 1 hour is required for replication of the chromosome. The ratio of the number of copies of genes near the replication origin and terminus is different than if 2 hours were required for replication of the DNA.

Instead of measuring numbers of copies of genes by transduction frequencies, the more precise method of DNA hybridization could be used because the locations of the origin and terminus were known. DNA was extracted from the cells, denatured, and immobilized on filter paper. Then an excess of a mixture of 3H-labeled DNA fragment from the origin and 14C-labeled DNA fragment from the terminus region was added and allowed to hybridize to the single-stranded DNA on the filters. When all hybridization to the immobilized DNA was completed, the filters were washed free of unannealed DNA and the ratio of bound 3H to 14C radioactivity was determined by liquid scintillation counting. This ratio reflects the ratio of the number of origins and termini in the culture of cells from which the DNA was extracted. The ratio could be measured using DNA extracted from cells growing at various growth rates. It showed that for cell doubling times in the range of 20 minutes to 3 hours the chromosome doubling time remained constant at about 40 minutes.

The constancy of the chromosome doubling time raises new problems, however. How do the cells manage to keep DNA replication and cell division precisely coordinated, and how can DNA replication, which requires 40 minutes, manage to keep up with cell division if cells are dividing in less than 40 minutes?