Molecular features of imprinted genes include differences in expression, me
thylation, and the timing of DNA replication between parental alleles. Wher
eas methylation differences always seem to be associated with differences i
n expression, differences in the timing of replication between parental hom
ologs are not always seen at imprinted loci. These observations raise the p
ossibility that differences in replication timing may not be an essential f
eature underlying genomic imprinting. In this study we examined the timing
of replication of the two alleles of the imprinted RSVIgmyc transgene in in
dividual embryonic cells using fluorescence in situ hybridization (FISH). T
he cis-acting signals for RSVIgmyc imprinting are within RSVIgmyc itself. T
hus, allele-specific differences in replication, if they indeed govern RSVI
gmyc imprinting, should be found in RSVIgmyc sequences. We found that the p
arental alleles of RSVIgmyc, which exhibit differences in methylation, repl
icated at the same time. Synchronous replication was also seen in embryonic
cells containing a modified version of RSVIgmyc that exhibited parental al
lele differences in both methylation and expression. These findings indicat
e that maintenance of expression and methylation differences between allele
s does not require a difference in replication timing. The differences in r
eplication timing of endogenous imprinted alleles detected by FISH might th
erefore reflect structural differences between the two alleles that could b
e a consequence of imprinting or, alternatively, could be unrelated to impr
inting. Dev. Genet. 23:000-000, 1998. (C) 1998 Wiley-Liss, Inc.