DNA replication initiation sites and initiation frequencies over 12.5 kb of
the human c-myc locus, including 4.6 kb of new 5' sequence, were determine
d based on short nascent DNA abundance measured by competitive polymerase c
hain reaction using 21 primer sets. In previous measurements, no comparativ
e quantitation of nascent strand abundance was performed, and distinction o
f major from minor initiation sites was not feasible. Two major initiation
sites were identified in this study. One predominant site has been located
at similar to 0.5 kb upstream of exon 1 of the c-myc gene, and a second new
major site is located in exon 2. The site in exon 2 has not been previousl
y identified. In addition, there are other sites that may act as less frequ
ently used initiation sites, some of wh ich may correspond to sites in prev
ious reports. Furthermore, a comparison of the abundance of DNA replication
intermediates over this same region of the c-myc locus between HeLa and no
rmal skin fibroblast (NSF) cells indicated that the relative distribution w
as very similar, but that nascent strand abundance in HeLa cells was approx
imately twice that in NSF relative to the abundance at the lamin B2 origin.
This increased activity at initiation sites in the c-myc locus may mainly
be influenced by regulators at higher levels in transformed cells like HeLa
. (C) 2000 Wiley-Liss, Inc.