Figure 2.

Mapping of the origin/terminus of replication by slow-moving Y-like arcs. Panels 1 and 2—diagrammatic explanation of patterns of 2D-AGE arcs resulting from ribonucleotide blockage of restriction sites. 1A and 2A: a uniform replication fork (Y) arc results when a replisome enters one end of a restriction fragment (length i) and proceeds to the other end without impediment; arrow indicates direction of replication. 1B and 2B: Where a restriction site is blocked by ribonucleotide incorporation on one strand (denoted by a cross), the adjacent fragment (length ii) remains joined, creating a slow-moving Y-like arc, SMY. 1C–D and 2C–D: If the adjacent fragment contains a replication terminus (ter) or unidirectional origin (ori), then the resulting series of replication intermediates will contain two junctions, one active, the other static. These form a distinct type of ‘flying' slow-moving Y-like arc (fSMY), whose ends do not meet the diagonal of linear molecules. The length of the static branch (iii) relative to that of the fragment (ii) to which it is joined determines how far above the linear diagonal the fSMY arc lies; arc 2D starts and ends well above the linear diagonal, whereas fSMY arc 2C lies much closer to it. Panels 3–8: 2D-AGE analysis of chick liver mtDNA digested with NheI, XhoI, MfeI, SalI, HincII and MscI, respectively; nucleotide coordinates define the unit-length linear fragment detected in each case. A representative intermediate of each of the fSMY arcs is depicted beneath the gel images. Crosses are blocked restriction sites; arrows indicate direction of movement of the growing fork, the other fork is static; gray-filled box—NCR. Radiolabeled probes used to detect mtDNA fragments are indicated at the top left of panels 3–8, and their approximate location is indicated on the schematic map at the base of the figure, together with relevant restriction sites. HincII and MscI also cut elsewhere, but only sites relevant to the arcs detected are shown.