The Masters and Broda experiment was designed to locate the origin of replication on the genetic map. It is primarily a genetic experiment, but it utilizes the fact discussed in the first chapter that an exponentially growing population contains more young individuals than old individuals. Chromosomes can be considered in the same way. Correspondingly, a population of growing and dividing chromosomes contains more members just beginning replication than members just finishing replication. The Cairns autoradiograph experiments show that the chromosome is replicated sequentially. A population of cells or chromosomes that is growing exponentially will contain more copies of genes located near the origin of replication than genes located at the terminus of replication. This same idea was used to locate the replication origin and demonstrate bidirectional replication of the SV40 animal virus.

Determining whether the bacterial chromosome is replicated in one direction from a unique origin or in both directions from a unique origin becomes a question of counting gene copies. If we could count the number of copies of genes A, B, C, D, and E, we could determine whether the cell uses monodirectional or bidirectional DNA replication initiating from point X (Fig. 1).

Fig1. Left: Monodirectional replication from point X generates a step plus gradient of marker frequencies. Right: Bidirectional replication originating from X generates two gradients of marker frequencies. In the drawings, successive initiations have taken place on the single chromosome.

Several methods exist for counting the relative numbers of copies of different genes or chromosome regions. Here we shall consider a bio logical method for performing such counting that utilizes the phage P1. This method depends upon the fact that after P1 infection, a cell synthesizes about 100 new P1 particles. Most of these package their own DNA. A few phage particles package E. coli DNA instead. If a P1 lysate that was prepared on one type of cells is then used to infect a second culture of cells, most of the infected cells will proceed to make new phage P1. Those few cells that are infected with a P1 coat containing E. coli DNA from the first cells may be able to recombine that particular stretch of E. coli DNA into their chromosomes. By this means they can replace stretches of chromosomal DNA with chromosomal DNA brought into them by the phage particles (Fig. 2). This process is termed transduction.

Fig2. Transduction of genetic markers by phage P1 infecting an X⁺ cell and carrying the X⁺ marker into an X^- cell.

The numbers of these defective phage particles carrying different genes from the infected cells is related to the numbers of copies of these genes present at the time of phage infection. Transduced cells can be made to reveal themselves as colonies, thereby permitting their simple and accurate quantitation. Consequently the use of phage P1 enabled counting of the relative numbers of copies within growing cells of various genes located around the chromosome. The results together with the known genetic map indicated that E. coli replicates its chromosome bidirectionally and determined the genetic location of the replication origin.

On the opposite side of the chromosome from the origin lies a terminus region. A protein called Tus binds to the terminus. It blocks elongation by inactivating the oncoming helicase of the replication fork.

Autoradiographic experiments have also suggested that mammalian DNA also replicates bidirectionally from origins (Fig. 3). Multiple replication forks or “eyes” could be shown to derive from a single DNA duplex. The replication trails indicated that two forks originated from a single origin. Furthermore, the rate of elongation of labeled strands showed that the synthesis rate of mammalian DNA is about 200 nucleotides per second.

Fig3. Schematic of an electron micrograph of an autoradiograph pre pared from mouse DNA extracted after 30 seconds of labeling with radioactive thymidine. DNA is replicated bidirectionally from the multiple origins.