II. REPLICATION OF DNA IN CHROMOSOMES 93 



stages of the cell cycle. Pei'haps the clearest case is the breakage of 

 chromosomes in the prophase stages in Lilmm after a demonstration 

 of DNA replication is no longer possible. These breaks produce chroma- 

 tid bridges in abundance (Mitra, 1958). Other examples are the produc- 

 tion of chromatid exchanges in pollen tubes and in the late interphase 

 in roots. Breaks that are produced before DNA replication in the G-^ 

 stage also undergo reunion or exchange befoi'e i-eplication. This is 

 indicated by the yield of chromosome type aberrations in some cells and 

 perhaps more convincingly by the results of variations of intensity and 

 fractionation of dose in radiation studies (Sax, 1939; Wolff and Luippold, 

 1956a). The yield of one-hit aberrations, i.e., single breaks, is linear with 

 dose, but exchanges that require interaction between ends produced from 

 separate breaks increase almost as the square of the dose. If the dose is 

 given over a longer period so that most breaks are repaired before 

 another is produced close enough for interaction, the curve of yield to 

 dose approaches linearity. On the other hand, one-hit aberrations show 

 no change with variations in rate, but are proportional to total dose. 

 There is also a decrease of the two-hit type when the dose is given in 

 two fractions separated by an interval long enough for nearly complete 

 healing of lesions. Wolff and Luippold (1956b) obtained evidence for 

 two kinds of aberrations in Vicia roots, a group that undergoes reunion 

 in about 1 minute and a second group that rejoins within 1 to 2 hours. 

 Since these experiments were carried out with Vicia seeds soaked for 

 18 to 24 hours in water, many of the cells are presumably in G^. Exposing 

 the cells to tritiated thymidine revealed no DNA synthesis (Wolff, 

 1960). These results indicate that broken chromosomes can rejoin before 

 DNA i-eplication. By exposing the roots to chloramphenicol (300/xg/ml) 

 the incorporation of glycine-C^* was reduced by 13-33%. When the 

 chloramphenicol was given before or after a single dose of radiation it 

 had no effect on aberrations, but when given for the period between two 

 doses it prevented the usual split dose effect, i.e., the breaks from the 

 first dose remained open and capable of exchanging with those produced 

 by the second. Aureomycin had a similar effect, but penicillin did not 

 prevent rejoining. On the basis of these experiments Wolff (1960) 

 maintains that protein synthesis is necessary for chromosome reunion 

 and supposes that the linear structure is maintained by protein. Since 

 DNA replication could not be demonstrated during the period of 

 rejoining, he assumes DNA is not involved. 



However, recent experiments (Taylor et al., 1962) indicate that DNA 

 replication may be involved in chromosome reunion. Fluorodeoxyuridy- 

 late is a potent inhibitor of thymidylate synthetase. When fluorodeoxy- 

 uridine (FUDR) is presented to cells, many of tlu>m ha\-e enzymes for 



