Molecular Genetics

Facts

• Total nuclear DNA from an asynchronously growing population of cells was digested with a restriction enzyme.
• The fragments were separated by two-dimensional gel electrophoresis. In the first dimension, separation was by mass. In the second dimension, the separation (high % agarose and high voltage) was also sensitive to the shapes of the molecules.
• The separated fragments were Southern blotted to a membrane. It was probed by hybridization repeatedly with a series of cloned genome fragments.
• Restriction fragments lacking a replication start site should form a series of Y-shaped molecules. Those containing start sites should have a bubble until one of the forks passes the restriction site at which digestion occurred. After that, the molecules should be Y-forms.
• Some probes revealed one hybridizing arc. Others revealed two hybridizing regions. Still others revealed a mixture of both patterns.
• Replicating fragments migrating as discontinuous regions are spaced, on average, 40 kbp from one another.
• The mixed pattern of hybridization is typical of the tandem ribosomal DNA repeats.

Interpretations

• The continuous arced pattern is due to Y-shaped molecules.
• The discontinuous pattern is due to segments containing replication start sites, the smaller molecules containing bubbles, the larger ones being Y-shaped. The discontinuity reflect the point that the fork reaches the end of the restriction fragment (i.e. the bubble bursts).
• That some probes reveal mixtures of the two patterns suggests that the start site contained in the assayed fragment was not functional in all cells. In the case of rDNA repeats, not all copies appeared to function as origins.

Further information

• In the S. cerevisiae genome, experiments such as these reveal specific start site locations. In the human genome, these experiments reveal regions in which replication begins without identifying specific start points (ref).
• There are other methods of identifying origins.

This is page 1322 of Molecular Genetics by Ulrich Melcher, © 1997, 1998, 1999