Developmental Regulation of the Tetrahymena thermophila Origin Recognition Complex
Figure 7
Two-dimensional gel analysis of rDNA replication intermediates during development.
(A) Schematic of the palindromic rDNA 5′NTS fragment generated by HindIII (H3) digestion, and possible replication intermediate patterns resolved by neutral-neutral 2D gel electrophoresis. Pro, promoter; D1 and D2, imperfect 430 bp tandem duplications and harbor the rDNA origins of replication. Simple Y (arrow), passive replication of 5′ NTS origins. Bubble (filled arrowhead) or bubble to Y, bidirectional replication within the 5′ NTS. Composite (simple Y arc and bubbles), active and passive replication of the 5′ NTS. Double Y, two converging forks initiating within or outside the 5′ NTS. Barrier, replication of the 5′ NTS by converging forks, in which the first fork entered and terminated at a barrier prior to entry of the second fork. Aberrant Y (unfilled arrowhead), simple Y arc containing partially replicated DNA. Diagonal dashed line, migration of linear duplex DNA fragments; dotted arc, reference pattern for simple Y arc intermediates. (B) C3 rDNA amplification replication intermediates after mating for 14 and 16 h, and subsequent refeeding (at 24 h) for an additional 8 h. Mating: strains SB4202 and SB1934. (C) Mung bean nuclease (MBN) digestion on C3 rDNA amplification intermediates in matings between SB4202 and SB1934 collected after refeeding with media for 8 h. DNA samples from log phase SB4204 stain were collected as the control.