508 R. E. HANDSCHUMACHER AND A. D. WELCH 



The most prominent aspect of the metabolism of these halogenated py- 

 rimidines is their remarkable capacity to replace thymine residues in DNA. 

 This substitution was first noted with bromouracil in S. faecalis m and has 

 been confirmed in E. coli, i2b • 426 E. coli T2 phage, and numerous other micro- 

 organisms 4 - 7 in which, under certain conditions, over 50% of the thymine 

 of the DNA can be replaced by this analog. Exposure of H.Ep. # 1 cells in 

 culture to bromodeoxyuridine resulted in up to 45 % replacement of thy- 

 midine by this analog with concomitant inhibition of growth. 4 - 8 With T2r + 

 phage in E. coli it was reported 429 that close to 100% of the thymine could 

 be replaced by bromouracil and yet 9% of the infectivity remained. This 

 result, coupled with the findings with HeLa cells and the stimulation of 

 bacterial growth by these analogs, strongly suggests that bromouracil can 

 replace thymine with retention of the functional activity of the DNA. How- 

 ever, the DNA obtained from E. coli grown in the presence of bromouracil 

 showed a heterogeneous distribution of the analog in the different molecular 

 species separated by chromatography on Ecteola columns. 430 In general, 

 the fractions containing bromouracil appeared more acidic in nature, a 

 finding consistent with the significantly lower pKa of bromouracil as com- 

 pared to that of thymine. Similarly, the DNA from T4 phage of E. coli iso- 

 lated after exposure to bromouracil exhibited marked heterogeneity when 

 subjected to equilibrium centrif ligation, in contrast to the normally ho- 

 mogeneous pattern displayed by DNA from untreated phage. 431 The differ- 

 ences in sedimentation rates were explicable on the basis of the difference 

 between the molecular weight of the methyl group and that of the bromine 

 atom (79.9); the heterogeneity was confirmed, as with the DNA from E. 

 coli, by chromatographic techniques. Unlike the situation with RNA for- 

 mation in cells exposed to azaguanine, it does not appear that bromouracil 

 results in the synthesis of excess "nonfunctional" DNA. 432 Positive identi- 

 fication of the deoxyribonucleotides of bromouracil, chlorouracil and iodo- 

 uracil following enzymic digestion of DNA from E. coli exposed to each 

 of these analogs, respectively, established the nature of the incorporation, 



426 D. B. Dunn and J. D. Smith, Nature 174, 305 (1954). 



426 S. Zamenhof and G. Griboff, Nature 174, 306 (1954). 



427 A. Wacker, A. Trebst, I). Jacherts, and F. Weygand, Z. Naturforsch. 9b, 616 

 (1954). 



428 M. L. Eidinoff, L. Cheong, and M. A. Rich, Federation Proc. 18, 220 (1959). 



429 R. M. Litman and A. B. Pardee, Nature 178, 529 (1956). 



430 A. Bendich, H. B. Pahl, and G. B. Brown, in "The Chemical Basis of Heredity" 

 W. I). McElroy and B. Glass, eds.), p. 378. Johns Hopkins Press, Baltimore, 

 Maryland, 1957. 



431 M. Meselson, F. W. Stahl, and J. Vinograd, Proc. Natl. Acad. Sci. U. S. 43, 581 

 (1957). 



432 S. Zamenhof, B. Reiner, R. DeGiovanni, and K. Rich, J. Biol. Chem. 219, 165 

 (1956). 



