Effect of the −1C>T polymorphism on the Kozak ANV sequence in an in vitro cell-free transcription/translation system.
(A) Representative autoradiogram of the SDS-PAGE analysis. A main band of 40 kDa is present both in −1C and in −1T plasmids, but not in the negative control. Densitometric analysis of ANV band shown in the gel (arbitrary units) indicates that more protein was synthesized from the −1T plasmid. (B) The 35S-methionine incorporated into acid-precipitable proteins in translation reactions containing −1C or −1T cDNA constructs of ANV. Results are mean (± SE) from 3 different series of experiments, each one including translation reactions with −1C or −1T cDNA or, as negative control, without cDNA. For each reaction, 6 replicates were analyzed, and the coefficient of variation between replicates was less than 30%. Precipitable radioactivity from negative control reaction without DNA was considered to be background and subtracted from corresponding values for −1C and −1T reactions. Significantly higher translation efficiency was associated with the −1T cDNA form (*P < .01).
